Methods and Compositions for Producing Alkenes of Various Chain Length

ABSTRACT

The NonA alkene synthase in  Synechococcus  sp. displays selective synthesis of 1-nonadecene. Heterologous recombination of a domain, i.e. the acyl binding domain, with other acyl binding proteins, affects acyl substrate chain-length binding selectivity and therefore the chain-length of the synthesized 1-alkenes. Compositions and methods are provided to selectively synthesize 1-alkenes of various chain lengths.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to earlier filed U.S. Ser. No.13/370,654, filed Feb. 10, 2012 (pending), and U.S. Provisional PatentApplication No. 61/441,619, filed Feb. 10, 2011, each of which is hereinincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 10, 2012, isnamed 20073US.txt and is 368,275 bytes in size.

BACKGROUND OF THE INVENTION

Unsaturated linear hydrocarbons such as α-olefins or 1-alkenes are anindustrially important group of molecules which can serve as feedstocksfor producing various materials such as detergents, fuels,pharmaceutical products, plastics, synthetic rubbers and viscosityadditives. Olefins or alkenes are unsaturated hydrocarbons whosemolecules contain one or more pairs of carbon atoms linked together by adouble bond.

Shorter alkene products are desirable in industry because of theirusefulness as surfactants and lubricants. Because 1-alkenes arehydrocarbons, they can also serve as fuels. In this context, 1-alkeneswith shorter carbon chain lengths are also preferred because they havelower melting points (FIG. 1). Thus, a need exists for improved methodsand compositions for synthesizing 1-alkenes of desired chain lengths.

SUMMARY OF THE INVENTION

The invention described herein relates to compositions and methods forsynthesizing 1-alkenes with defined chain lengths. In one embodiment,the disclosure provides alkene synthases that are modified such that theresulting chain length of the primary alkene product is different thanthe primary product produced by the unmodified or native alkenesynthase. For example, an alkene synthase that produces primarilynonadecene can be modified to produce primarily shorter alkenes, e.g.,heptadecene, tridecene, pentadecene, etc.

The present disclosure provides an isolated or recombinant NonA alkenesynthase comprising a heterologous acyl binding pocket. In oneembodiment, the heterologous acyl binding pocket comprises a polypeptidesequence of SEQ ID NO: 8. In another embodiment, the heterologous acylbinding pocket comprises a polypeptide sequence of SEQ ID NO: 12. Instill another embodiment, the heterologous acyl binding pocket comprisesa polypeptide sequence of SEQ ID NO: 16. In further embodiments, theheterologous acyl binding pocket comprises a polypeptide sequence atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 8, SEQID NO: 12, or SEQ ID NO: 16.

The present disclosure also provides an isolated or recombinantpolynucleotide encoding a heterologous acyl binding pocket. In oneaspect, the nucleotide sequence encoding the heterologous acyl bindingpocket comprises SEQ ID NO: 35. In another aspect, the nucleotidesequence encoding the heterologous acyl binding pocket comprises SEQ IDNO: 36. In yet another aspect, the nucleotide sequence encoding theheterologous acyl binding pocket comprises SEQ ID NO: 34. In oneembodiment, the nucleotide sequence encoding the heterologous acylbinding pocket comprises a nucleotide sequence that is a degeneratevariant of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34. In anotherembodiment, the nucleotide sequence encoding the heterologous acylbinding pocket comprises a nucleotide sequence that is at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% or at least 99.9% identical to SEQID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34. In yet another embodiment,the nucleotide sequence encoding the heterologous acyl binding pocketcomprises a nucleotide sequence that encodes a polypeptide at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%%, at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%,at least 99.8% or at least 99.9% identical to SEQ ID NO: 8, SEQ ID NO:12, or SEQ ID NO: 16. In still another embodiment, the nucleotidesequence encoding the heterologous acyl binding pocket comprises anucleotide sequence that hybridizes under stringent conditions to SEQ IDNO: 35, SEQ ID NO: 36, or SEQ ID NO: 34.

The invention relates to an isolated or recombinant polypeptide encodinga chimeric alkene synthase comprising or consisting of an amino acidsequence SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29. In oneembodiment, the polypeptide sequence is at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% orat least 99.9% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO:29.

The present disclosure provides an isolated or recombinantpolynucleotide encoding a chimeric alkene synthase comprising orconsisting of a nucleotide sequence selected from the group consistingof SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 26. In one embodiment,the nucleotide sequence is a degenerate variant of SEQ ID NO: 27, SEQ IDNO: 28, or SEQ ID NO: 26. In another embodiment, the nucleotide sequenceis at least 71%, at least 72%, at least 73%, at least 74%, at least 75%,at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99% or at least 99.9%identical to SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26. In stillanother embodiment, the nucleotide sequence encodes a polypeptide havingthe amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO:29. In yet another embodiment, the nucleotide sequence encodes apolypeptide at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, atleast 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29. In one aspect, thenucleotide sequence hybridizes under stringent conditions to a nucleicacid sequence that encodes a polypeptide having the amino acid sequenceof SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26.

In one aspect, the isolated polynucleotide of the invention is operablylinked to one or more expression control sequences. In another aspect, avector is provided, wherein said vector comprises an isolatedpolynucleotide described herein. In yet another aspect, a fusion proteincomprising the isolated polypeptide is fused to a heterologous aminoacid sequence is provided.

In one embodiment, the invention provides a host cell comprising one ormore isolated polynucleotides described herein. In a further embodiment,the host cell is a photoautotroph. In another further embodiment, thehost cell is E. coli. In another embodiment, the host cell is aprokaryote, a eukaryote, a yeast, a filamentous fungus, a protozoa, analgae, or a synthetic cell. In yet another embodiment, the host cellproduces a carbon-based product of interest. Also provided is anisolated antibody or antigen-binding fragment or derivative thereofwhich binds selectively to an isolated polypeptide described herein.

The present disclosure also provides methods for producing carbon-basedproducts of interest, comprising: culturing a host cell to produce thecarbon-based product of interest, wherein the host cell comprises arecombinant nucleotide sequence encoding a chimeric alkene synthasecomprising a heterologous acyl binding pocket; and isolating thecarbon-based product of interest. In another embodiment, the chimericalkene synthase is an engineered NonA protein. In a further embodiment,the NonA comprises SEQ ID NO: 2. In another further embodiment, the NonAcomprises SEQ ID NO: 24. In still another embodiment, the heterologousacyl binding pocket comprises the amino acid sequence SEQ ID NO: 8, SEQID NO: 12, or SEQ ID NO: 14. In still another embodiment, the chimericalkene synthase selectively synthesizes an alkene with a specific chainlength. In a further embodiment, the synthesized alkene is a propene, abutene, a pentene, a heptene, an octene, a nonene, a decene, anundecene, a dodecane, a tridecene, a tetradecene, a pentadecene, ahexadecene, a heptadecene, an octadecene, a nonadecene, an eicosene, anuneicosene, or a doeicosene, or isomers and mixtures thereof. In yetanother embodiment, the synthesized alkene is 1-tridecene,1-pentadecene, 1, heptadecene, or 1-nonadecene.

In yet another embodiment, a method is provided for identifying amodified alkene synthase gene that selectively catalyzes the formationof a desired alkene, comprising: modifying an alkene synthase byreplacing the acyl carrier binding domain with a heterologous acylcarrier binding domain; expressing the modified alkene synthase in ahost cell; and screening the host cell for production of the selectedalkene. Also provided is an improved alkene synthase enzyme identifiedby the above method.

In one aspect, a method for producing a carbon-based product of interestis provided, comprising the steps of: culturing a host cell to producethe carbon-based product of interest, wherein the host cell comprises anengineered chimeric NonA comprising a heterologous binding pocket; andisolating the carbon-based product of interest. In a further aspect, thechimeric alkene synthase selectively synthesizes one or more alkeneswith specific chain lengths. In yet another further aspect, the one ormore alkenes are selected from the group consisting of: propene, butene,pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene,tridecene, tetradecene, pentadecene, hexadecene, heptadecene,octadecene, nonadecene, eicosene, uneicosene, doeicosene, and isomersand mixtures thereof. In yet another embodiment, the synthesized alkeneis 1-tridecene, 1-pentadecene, 1, heptadecene, or 1-nonadecene.

In one embodiment, a method for producing a tridecene or pentadecene isprovided, comprising the steps of: culturing a host cell to produce thetridecene or pentadecene, wherein the host cell comprises an engineeredchimeric NonA comprising a heterologous SafB binding pocket (SEQ ID NO:8); and isolating the tridecene or pentadecene. In another embodiment, amethod for producing a heptadecene is provided, comprising the steps of:culturing a host cell to produce the heptadecene, wherein the host cellcomprises an engineered chimeric NonA comprising a heterologous MycAbinding pocket (SEQ ID NO: 12); and isolating the heptadecene. In stillanother embodiment, a method for producing a heptadecene is provided,comprising the steps of: culturing a host cell to produce theheptadecene, wherein the host cell comprises an engineered chimeric NonAcomprising a heterologous DptE binding pocket (SEQ ID NO: 16); andisolating the heptadecene.

In still another embodiment, a method for producing a nonadecene orheptadecene is provided, comprising the steps of: culturing a host cellto produce the nonadecene or heptadecene, wherein the host cellcomprises an engineered NonA (SEQ ID NO: 24); and isolating thenonadecene or heptadecene.

Additional information related to the invention may be found in thefollowing Drawings and Detailed Description.

DRAWINGS

FIG. 1 provides melting and boiling points of alkenes with various chainlengths.

FIG. 2 is a representation of the domains found in the 1-alkene synthaseYP_(—)001734428 (NonA), as identified by the conserved domain (CD)searching program available on the NCBI website. Abbreviations fordomains: acyl-carrier protein (ACP); phosphopantetheinyl (PP);ketosynthase (KS); acyltransferase (AT); ketoreductase (KR);sulfotransferase (ST); and thioesterase (TE). By reference to theYP_(—)001734428 gene sequence, the domains are located at the followingresidues: LuxE domain: 10-557; ACP domain: 598-675; KS domain: 693-1095;AT domain: 1216-1490; KR domain: 1777-1943; ST domain: 2145-2360; TEdomain: 2449-2708.

FIG. 3 illustrates the putative mechanism of 1-nonadecene biosynthesisfrom stearic acid, stearyl-ACP or stearyl-CoA. AT, acyltransferase; ACP,acyl-carrier protein; KS, ketosynthase; KR, ketoreductase; ST,sulfotransferase; TE, thioesterase.

FIG. 4A-B is a representation of the residues of the acyl binding domainof saframycin M×1 synthetase B complexed with an acyl-adenylate ligand.FIG. 4(A) The residues of the acyl binding pocket of the saframycin M×1synthetase B acyl-transferase are shown surrounding thedodecanoyl-ligand (white). The end of the acyl chain of the ligand isindicated. FIG. 4(B) The residues of the binding pocket which are notstrictly conserved between the four acyl binding pockets are show inblack while the others are shown in grey. “*” indicates Cys324.

FIG. 5 is an amino acid alignment of acyl ligase domains of NonA (SEQ IDNO: 40), DptE (SEQ ID NO: 14), MycA (SEQ ID NO: 39), and SafB (SEQ IDNO: 6). The interior acyl binding domain (IABD) of NonA, DptE, MycA, andSafB is underlined in black.

FIG. 6A-B provides representations of the interior acyl binding pocketof the SafB acyl ligase domain. FIG. 6(A) The amino acids of theinterior acyl binding pocket in the SafB acyl ligase domain are blackwhile the rest are grey. FIG. 6(B) View of the binding pocket with allresidues 5 angstroms or closer to the acyl-adenylate (white) indicated.The end of the acyl chain of the ligand is indicated.

FIG. 7 depicts a stack of GC/MS chromatograms comparing cell pelletextracts of JCC2157 and JCC308. The interval between the tick marks onthe MS detector axis is 1000.

FIG. 8A-D provides mass spectra of identified 1-alkenes in cellextracts. FIG. 8 (A) The MS fragmentation spectrum of the JCC21571-heptadecene peak plotted above the spectrum in the NIST database. FIG.8 (B) The MS fragmentation spectrum of the JCC2157 1-octadecene peakplotted above the spectrum in the NIST database. FIG. 8 (C) The MSfragmentation spectrum of the JCC2157 1-nonadecene peak plotted abovethe spectrum in the NIST database. FIG. 8 (D) The mass spectrum of theJCC2157 peak identified as 1,x-nonadecadiene (C19:2).

FIG. 9 shows the GC/MS chromatogram of the cell pellet extract ofJCC2375 plotted above the chromatogram of the cell pellet extract ofJCC2157. The interval between the tick marks on the MS detector axis is2000.

FIG. 10 represents the MS fragmentation spectrum of theJCC23751-tridecene peak plotted above the spectrum in the NIST database.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the invention shall have the meanings that are commonlyunderstood by those of ordinary skill in the art. Further, unlessotherwise required by context, singular terms shall include the pluraland plural terms shall include the singular. Generally, nomenclaturesused in connection with, and techniques of, biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are those wellknown and commonly used in the art.

The methods and techniques are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002); Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction toGlycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual,Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry:Section A Proteins, Vol. I, CRC Press (1976); Handbook of Biochemistry:Section A Proteins, Vol. II, CRC Press (1976); Essentials ofGlycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinter-nucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hair-pinned, circular, or in apadlocked conformation.

Unless otherwise indicated, and as an example for all sequencesdescribed herein under the general format “SEQ ID NO:”, “nucleic acidcomprising SEQ ID NO:1” refers to a nucleic acid, at least a portion ofwhich has either (i) the sequence of SEQ ID NO:1, or (ii) a sequencecomplementary to SEQ ID NO:1. The choice between the two is dictated bythe context. For instance, if the nucleic acid is used as a probe, thechoice between the two is dictated by the requirement that the probe becomplementary to the desired target.

An “isolated” RNA, DNA or a mixed polymer is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases and genomic sequences with which it is naturally associated.

As used herein, an “isolated” organic molecule (e.g., an alkane, alkene,or alkanal) is one which is substantially separated from the cellularcomponents (membrane lipids, chromosomes, proteins) of the host cellfrom which it originated, or from the medium in which the host cell wascultured. The term does not require that the biomolecule has beenseparated from all other chemicals, although certain isolatedbiomolecules may be purified to near homogeneity.

The term “recombinant” refers to a biomolecule, e.g., a gene or protein,that (1) has been removed from its naturally occurring environment, (2)is not associated with all or a portion of a polynucleotide in which thegene is found in nature, (3) is operatively linked to a polynucleotidewhich it is not linked to in nature, or (4) does not occur in nature.The term “recombinant” can be used in reference to cloned DNA isolates,chemically synthesized polynucleotide analogs, or polynucleotide analogsthat are biologically synthesized by heterologous systems, as well asproteins and/or mRNAs encoded by such nucleic acids.

As used herein, an endogenous nucleic acid sequence in the genome of anorganism (or the encoded protein product of that sequence) is deemed“recombinant” herein if a heterologous sequence is placed adjacent tothe endogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. In this context, aheterologous sequence is a sequence that is not naturally adjacent tothe endogenous nucleic acid sequence, whether or not the heterologoussequence is itself endogenous (originating from the same host cell orprogeny thereof) or exogenous (originating from a different host cell orprogeny thereof). By way of example, a promoter sequence can besubstituted (e.g., by homologous recombination) for the native promoterof a gene in the genome of a host cell, such that this gene has analtered expression pattern. This gene would now become “recombinant”because it is separated from at least some of the sequences thatnaturally flank it.

A nucleic acid is also considered “recombinant” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “recombinant” if it contains an insertion, deletion or apoint mutation introduced artificially, e.g., by human intervention. A“recombinant nucleic acid” also includes a nucleic acid integrated intoa host cell chromosome at a heterologous site and a nucleic acidconstruct present as an episome.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence. The term “degenerate oligonucleotide” or “degenerate primer”is used to signify an oligonucleotide capable of hybridizing with targetnucleic acid sequences that are not necessarily identical in sequencebut that are homologous to one another within one or more particularsegments.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990) (hereby incorporated by reference in itsentirety). For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1, hereinincorporated by reference. Alternatively, sequences can be comparedusing the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

A particular, non-limiting example of a mathematical algorithm utilizedfor the comparison of sequences is that of Karlin and Altschul (Proc.Natl. Acad. Sci. (1990) USA 87:2264-68; Proc. Natl. Acad. Sci. USA(1993) 90: 5873-77) as used in the NBLAST and XBLAST programs (version2.0) of Altschul et al. (J. Mol. Biol. (1990) 215:403-10). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST polypeptide searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to polypeptide molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (Nucleic Acids Research (1997)25(17):3389-3402). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used (http://www.ncbi.nlm.nih.gov). One skilled in the art mayalso use the ALIGN program incorporating the non-linear algorithm ofMyers and Miller (Comput. Appl. Biosci. (1988) 4:11-17). For amino acidsequence comparison using the ALIGN program one skilled in the art mayuse a PAM 120 weight residue table, a gap length penalty of 12, and agap penalty of 4.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, preferably at leastabout 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference.For purposes herein, “stringent conditions” are defined for solutionphase hybridization as aqueous hybridization (i.e., free of formamide)in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1%SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1%SDS at 65° C. for 20 minutes. It will be appreciated by the skilledworker that hybridization at 65° C. will occur at different ratesdepending on a number of factors including the length and percentidentity of the sequences which are hybridizing.

A preferred, non-limiting example of stringent hybridization conditionsincludes hybridization in 4× sodium chloride/sodium citrate (SSC), atabout 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. Apreferred, non-limiting example of highly stringent hybridizationconditions includes hybridization in 1×SSC, at about 65-70° C. (orhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Intermediate rangese.g., at 65-70° C. or at 42-50° C. are also within the scope of theinvention. SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA,pH 7.4) can be substituted for SSC (1×SSC is 0.15 M NaCl and 15 mMsodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m) (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41 (% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M).

The skilled practitioner recognizes that reagents can be added tohybridization and/or wash buffers. For example, to decrease non-specifichybridization of nucleic acid molecules to, for example, nitrocelluloseor nylon membranes, blocking agents, including but not limited to, BSAor salmon or herring sperm carrier DNA and/or detergents, including butnot limited to, SDS, chelating agents EDTA, Ficoll, PVP and the like canbe used. When using nylon membranes, in particular, an additional,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (Church andGilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995,) or,alternatively, 0.2×SSC, 1% SDS.

The nucleic acids (also referred to as polynucleotides) may include bothsense and antisense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. They may be modified chemicallyor biochemically or may contain non-natural or derivatized nucleotidebases, as will be readily appreciated by those of skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.) Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule. Other modifications caninclude, for example, analogs in which the ribose ring contains abridging moiety or other structure such as the modifications found in“locked” nucleic acids.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989)and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57(1988)).

The term “attenuate” as used herein generally refers to a functionaldeletion, including a mutation, partial or complete deletion, insertion,or other variation made to a gene sequence or a sequence controlling thetranscription of a gene sequence, which reduces or inhibits productionof the gene product, or renders the gene product non-functional. In someinstances a functional deletion is described as a knockout mutation.Attenuation also includes amino acid sequence changes by altering thenucleic acid sequence, placing the gene under the control of a lessactive promoter, down-regulation, expressing interfering RNA, ribozymesor antisense sequences that target the gene of interest, or through anyother technique known in the art. In one example, the sensitivity of aparticular enzyme to feedback inhibition or inhibition caused by acomposition that is not a product or a reactant (non-pathway specificfeedback) is lessened such that the enzyme activity is not impacted bythe presence of a compound. In other instances, an enzyme that has beenaltered to be less active can be referred to as attenuated.

A “deletion” is the removal of one or more nucleotides from a nucleicacid molecule or one or more amino acids from a protein, the regions oneither side being joined together.

A “knock-out” is a gene whose level of expression or activity has beenreduced to zero. In some examples, a gene is knocked-out via deletion ofsome or all of its coding sequence. In other examples, a gene isknocked-out via introduction of one or more nucleotides into itsopen-reading frame, which results in translation of a non-sense orotherwise non-functional protein product.

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC), fosmids, phageand phagemids. Another type of vector is a viral vector, whereinadditional DNA segments may be ligated into the viral genome (discussedin more detail below). Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g., vectorshaving an origin of replication which functions in the host cell). Othervectors can be integrated into the genome of a host cell uponintroduction into the host cell, and are thereby replicated along withthe host genome. Moreover, certain preferred vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”(or simply “expression vectors”).

“Operatively linked” or “operably linked” expression control sequencesrefers to a linkage in which the expression control sequence iscontiguous with the gene of interest to control the gene of interest, aswell as expression control sequences that act in trans or at a distanceto control the gene of interest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) exists in a purity not found in nature, wherepurity can be adjudged with respect to the presence of other cellularmaterial (e.g., is free of other proteins from the same species) (3) isexpressed by a cell from a different species, or (4) does not occur innature (e.g., it is a fragment of a polypeptide found in nature or itincludes amino acid analogs or derivatives not found in nature orlinkages other than standard peptide bonds). Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A polypeptide or protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.As thus defined, “isolated” does not necessarily require that theprotein, polypeptide, peptide or oligopeptide so described has beenphysically removed from its native environment.

An isolated or purified polypeptide is substantially free of cellularmaterial or other contaminating polypeptides from the expression hostcell from which the polypeptide is derived, or substantially free fromchemical precursors or other chemicals when chemically synthesized. Inone embodiment, an isolated or purified polypeptide has less than about30% (by dry weight) of contaminating polypeptide or chemicals, moreadvantageously less than about 20% of contaminating polypeptide orchemicals, still more advantageously less than about 10% ofcontaminating polypeptide or chemicals, and most advantageously lessthan about 5% contaminating polypeptide or chemicals.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide. In a preferredembodiment, the polypeptide fragment is a contiguous sequence in whichthe amino acid sequence of the fragment is identical to thecorresponding positions in the naturally-occurring sequence. Fragmentstypically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferablyat least 12, 14, 16 or 18 amino acids long, more preferably at least 20amino acids long, more preferably at least 25, 30, 35, 40 or 45, aminoacids, even more preferably at least 50 or 60 amino acids long, and evenmore preferably at least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002) (herebyincorporated by reference).

The terms “thermal stability” and “thermostability” are usedinterchangeably and refer to the ability of an enzyme (e.g., whetherexpressed in a cell, present in an cellular extract, cell lysate, or inpurified or partially purified form) to exhibit the ability to catalyzea reaction at least at about 20° C., preferably at about 25° C. to 35°C., more preferably at about 37° C. or higher, in more preferably atabout 50° C. or higher, and even more preferably at least about 60° C.or higher.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusions that include theentirety of the proteins have particular utility. The heterologouspolypeptide included within the fusion protein is at least 6 amino acidsin length, often at least 8 amino acids in length, and usefully at least15, 20, and 25 amino acids in length. Fusions that include largerpolypeptides, such as an IgG Fc region, and even entire proteins, suchas the green fluorescent protein (“GFP”) chromophore-containingproteins, have particular utility. Fusion proteins can be producedrecombinantly by constructing a nucleic acid sequence which encodes thepolypeptide or a fragment thereof in frame with a nucleic acid sequenceencoding a different protein or peptide and then expressing the fusionprotein. Alternatively, a fusion protein can be produced chemically bycrosslinking the polypeptide or a fragment thereof to another protein.

As used herein, the term “antibody” refers to a polypeptide, at least aportion of which is encoded by at least one immunoglobulin gene, orfragment thereof, and that can bind specifically to a desired targetmolecule. The term includes naturally-occurring forms, as well asfragments and derivatives.

Fragments within the scope of the term “antibody” include those producedby digestion with various proteases, those produced by chemical cleavageand/or chemical dissociation and those produced recombinantly, so longas the fragment remains capable of specific binding to a targetmolecule. Among such fragments are Fab, Fab′, Fv, F(ab′)₂, and singlechain Fv (scFv) fragments.

Derivatives within the scope of the term include antibodies (orfragments thereof) that have been modified in sequence, but remaincapable of specific binding to a target molecule, including:interspecies chimeric and humanized antibodies; antibody fusions;heteromeric antibody complexes and antibody fusions, such as diabodies(bispecific antibodies), single-chain diabodies, and intrabodies (see,e.g., Intracellular Antibodies: Research and Disease Applications (1998)Marasco, ed., Springer-Verlag New York, Inc.), the disclosure of whichis incorporated herein by reference in its entirety).

As used herein, antibodies can be produced by any known technique,including harvest from cell culture of native B lymphocytes, harvestfrom culture of hybridomas, recombinant expression systems and phagedisplay.

The term “non-peptide analog” refers to a compound with properties thatare analogous to those of a reference polypeptide. A non-peptidecompound may also be termed a “peptide mimetic” or a “peptidomimetic.”See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford UniversityPress (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: AHandbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—APractical Textbook, Springer Verlag (1993); Synthetic Peptides: A UsersGuide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med.Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veberand Freidinger, Trends Neurosci., 8:392-396 (1985); and references sitedin each of the above, which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar to usefulpeptides may be used to produce an equivalent effect and are thereforeenvisioned to be part of the invention.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild-type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein.

A mutein has at least 85% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having at least 90% overallsequence homology to the wild-type protein.

In an even more preferred embodiment, a mutein exhibits at least 95%sequence identity, even more preferably 98%, even more preferably 99%and even more preferably 99.9% overall sequence identity.

Sequence homology may be measured by any common sequence analysisalgorithm, such as Gap or Bestfit.

Amino acid substitutions can include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2^(nd) ed.1991), which is incorporated herein by reference. Stereoisomers (e.g.,D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α-, α-disubstituted amino acids, N-alkyl amino acids, andother unconventional amino acids may also be suitable components forpolypeptides. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similaramino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptidenotation used herein, the left-hand end corresponds to the aminoterminal end and the right-hand end corresponds to the carboxy-terminalend, in accordance with standard usage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-331 and 25:365-389(herein incorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using a measure of homology assignedto various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild-type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Preferred parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. (Pearson,Methods Enzymol. 183:63-98 (1990) (herein incorporated by reference).For example, percent sequence identity between amino acid sequences canbe determined using FASTA with its default parameters (a word size of 2and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereinincorporated by reference.

“Specific binding” refers to the ability of two molecules to bind toeach other in preference to binding to other molecules in theenvironment. Typically, “specific binding” discriminates overadventitious binding in a reaction by at least two-fold, more typicallyby at least 10-fold, often at least 100-fold. Typically, the affinity oravidity of a specific binding reaction, as quantified by a dissociationconstant, is about 10⁻⁷ M or stronger (e.g., about 10⁻⁸ M, 10⁻⁹ M oreven stronger).

“Percent dry cell weight” refers to a measurement of hydrocarbonproduction obtained as follows: a defined volume of culture iscentrifuged to pellet the cells. Cells are washed then dewetted by atleast one cycle of microcentrifugation and aspiration. Cell pellets arelyophilized overnight, and the tube containing the dry cell mass isweighed again such that the mass of the cell pellet can be calculatedwithin ±0.1 mg. At the same time cells are processed for dry cell weightdetermination, a second sample of the culture in question is harvested,washed, and dewetted. The resulting cell pellet, corresponding to 1-3 mgof dry cell weight, is then extracted by vortexing in approximately 1 mlacetone plus butylated hydroxytolune (BHT) as antioxidant and aninternal standard, e.g., n-heptacosane. Cell debris is then pelleted bycentrifugation and the supernatant (extractant) is taken for analysis byGC. For accurate quantitation of 1-alkene, flame ionization detection(FID) was used as opposed to MS total ion count. 1-alkene concentrationsin the biological extracts were calculated using calibrationrelationships between GC-FID peak area and known concentrations ofauthentic 1-alkene standards. Knowing the volume of the extractant, theresulting concentrations of the 1-alkenespecies in the extracant, andthe dry cell weight of the cell pellet extracted, the percentage of drycell weight that comprised 1-alkene can be determined.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

The term “substrate affinity” as used herein refers to the bindingkinetics, K_(m), the Michaelis-Menten constant as understood by onehaving skill in the art, for a substrate. Various chimeric alkenesynthases can have a higher substrate affinity for alkenes of a certainchain length, making them selective for these alkenes.

The term “carbon source” as used herein refers to inorganic carbon,exogenous sugar or biomass.

Inorganic carbon is carbon provided in a molecule that cannot itself bemetabolized for energy by an organism, such as CO₂, carbonic acid, andcarbonate. Sources of inorganic carbon include CO₂, air, carbonic acid,carbonate salts, and emissions such as flue gas.

Carbon dioxide (which, along with carbonic acid, bicarbonate and/orcarbonate define the term “inorganic carbon”) is converted in thephotosynthetic process to organic compounds. The inorganic carbon sourceincludes any way of delivering inorganic carbon, optionally in admixturewith any other combination of compounds which do not serve as theprimary carbon feedstock, but only as a mixture or carrier (for example,emissions from biofuel (e.g., ethanol) plants, power plants,petroleum-based refineries, as well as atmospheric and subterraneansources).

A reduced or organic carbon source is a carbon based molecule that canbe metabolized by an organism for energy such as, for example, acarbohydrate (including a sugar or polysaccharide), amino acid, protein,organic acid, fatty acid, lipid, acetyl CoA, or any biosyntheticprecursor of any of these biomolecules.

“Carbon-based products of interest” include alkenes such as propene,butene, pentene, hexene, heptene, octene, nonene, decene, undecene,dodecene, tridecene, tetradecene, pentadecene, hexadecene, heptadecene,octadecene, nonadecene, eicosene, uneicosene, doeicosene, and isomersand mixtures thereof.

A “biofuel” as used herein is any fuel that derives from a biologicalsource. Biofuel refers to one or more hydrocarbons (e.g., 1-nonadecene),one or more alcohols, one or more fatty esters or a mixture thereof.Preferably, liquid hydrocarbons are used.

As used herein, the term “hydrocarbon” generally refers to a chemicalcompound that consists of the elements carbon (C), hydrogen (H) andoptionally oxygen (O). There are essentially three types ofhydrocarbons, e.g., aromatic hydrocarbons, saturated hydrocarbons andunsaturated hydrocarbons such as alkenes, alkynes, and dienes. The termalso includes fuels, biofuels, plastics, waxes, solvents and oils.Hydrocarbons encompass biofuels, as well as plastics, waxes, solventsand oils.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used and will beapparent to those of skill in the art. All publications and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. The materials, methods, and examples areillustrative only and not intended to be limiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Nucleic Acid Sequences

Cyanobacteria are known to be producers of hydrocarbons (Lin et al.(1996) Bioch. Biophy. Res. Comm., 228: 764-773; Chang et al., (2004) J.Nat. Prod. 67: 1356-1367). WO/2011/005548, herein incorporated byreference, describes genes responsible for the production of 1-alkenesin Synechococcus sp. PCC 7002. Other long chain hydrocarbons are knownto be produced in related, but distinct, microorganisms, e.g.,Synechococcus sp. PCC 7942 (produces heptadecane), Synechocystis sp. PCC6803 (reported to produce heptadecane), Nostoc sp. PCC 7120 (producesheptadecane), Thermosynechococcus sp. BP-1 (produces heptadecane) andCyanothece sp. ATCC 51142 (produces pentadecane).

The 1-alkene synthase YP_(—)001734428 contains 7 domains which implicateit in the biosynthesis of 1-nonadecene (FIG. 2). A LuxE domain ispresent which indicates that the protein can attach a fatty acid byacting as an acyltransferase (AT). LuxE is the protein which serves asan acyl-protein synthetase in the Lux operon (Lin et al. (1996)). Aphosphopantetheinyl (PP) attachment site is next which is characteristicof acyl-carrier protein (ACP) domains present in polyketide synthases(i.e. alkene synthases). Several other domains characteristic ofpolyketide synthases are also present including: a ketosynthase (KS)domain; an acyltransferase (AT) domain; an NADP site which indicates aketoreductase (KR) domain; a sulfotransferase (ST) domain; and athioesterase (TE) domain.

The biosynthesis of alkenes is similar to polyketide biosynthesis, wherea thioester bond is formed between the acyl starter unit and the ACPdomain of the enzyme. A Claisen condensation catalyzed by aβ-ketosynthase (KS) occurs between the acyl-thioester substrate andmalonyl-CoA to extend the chain by two carbons. The β-carbonyl isreduced by the ketoreductase domain, and the sulfotransferase domainserves to attach a sulfonate to the β-hydroxy group to form a sulfateintermediate. The last step in the pathway is a decarboxylativeelimination of sulfate catalyzed by the thioesterase domain to yield theterminal alkene (FIG. 3). This mechanism of terminal alkene formationvia action of a sulfotransferase and thioesterase domain has beendemonstrated for the unrelated metabolite curacin A (Gu et al. 2009).

An object of the invention described herein is to express in a host cella gene encoding a chimeric alkene synthase which selectively binds to analkene precursor of a pre-defined carbon chain length in an alkenesynthesis pathway to produce 1-alkenes of chain length-specific alkenesand other carbon-based products of interest. The pathway and/or chimericalkene synthase can be over-expressed in a Synechococcus strain such asSynechococcus sp. PCC 7002 or expressed in any other photosyntheticorganism to produce a hydrocarbon from light and inorganic carbon. Itcan also be expressed in non-photosynthetic organisms to producehydrocarbons from sugar sources.

Accordingly, one embodiment provides isolated nucleic acid moleculesencoding proteins having alkene synthase activity and/or hydrolaseactivity, and variants thereof, including expression optimized forms ofacyl binding pockets, and methods of improvement thereon. Thefull-length nucleic acid sequence (SEQ ID NO: 1) for the alkene synthasegene from Synechococcus sp. PCC 7002, YP_(—)001734428, is providedherein, as is the protein sequence (SEQ ID NO: 2) (see FIG. 2). Alsoprovided herein are optimized coding sequences for the alkene synthasegene, nonA_optV6, encoded by the nucleotide sequence of SEQ ID NO: 23,and expressing the recombinant NonA_optV6 protein encoded by SEQ ID NO:24. Also provided herein is a coding (SEQ ID NO: 5) and amino acidsequence (SEQ ID NO: 6) for a saframycin M×1 synthetase from Legionellapneumophila, a coding (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO:10) for a mycosubtilin synthase from Bacillus subtilis, and a coding(SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 14) for an acyl-CoAligase from Streptomyces filamentosus. Also provided herein aresequences of acyl binding pocket alignments of the above genes, andchimeric forms of the full-length nucleic acid sequence.

In addition, one embodiment provides a chimeric alkene synthaseconsisting of the Synechococcus sp. PCC 7002 NonA alkene synthase with aheterologous acyl binding pocket replacing the native binding pocket. Inone embodiment, the heterologous binding pocket may be selected from thegroup consisting of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. Inanother embodiment, the heterologous binding pocket may be selected fromthe group consisting of SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 29.Locations for insertion of the heterologous binding pocket into the NonAalkene synthase gene for one embodiment are provided. In otherembodiments, the heterologous binding pocket is inserted into the NonAalkene synthase gene in a region comparable to the native heterologousbinding pocket region location i.e., less than 5 peptides, less than 10peptides, less than 20 peptides, less than 50 peptides, less than 75peptides, less than 100 peptides, less than 150 peptides, or less than200 peptides upstream or downstream from the location of the nativebinding pocket region. The invention also includes nucleic acidsencoding the above-mentioned chimeric alkene synthases.

One embodiment provides an isolated nucleic acid molecule having anucleic acid sequence comprising or consisting of a chimeric alkenesynthase gene homologs, variants and derivatives of the chimeric alkenesynthase selected from the gene coding sequences SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.Another embodiment provides nucleic acid molecules comprising orconsisting of sequences which are structurally and functionallyoptimized versions of the chimeric alkene synthase gene. In a preferredembodiment, nucleic acid molecules and homologs, variants andderivatives comprising or consisting of sequences optimized forsubstrate affinity and/or substrate catalytic conversion rate areprovided.

A further embodiment provides nucleic acid molecules and homologs,variants and derivatives thereof comprising or consisting of sequenceswhich are variants of the chimeric NonA gene having at least 90%identity to SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO: 26, SEQID NO: 27, and SEQ ID NO: 28. Another embodiment provides nucleic acidmolecules and homologs, variants and derivatives comprising orconsisting of sequences which are variants of the chimeric alkenesynthase gene having at least 90% identity to SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 andoptimized for substrate affinity, substrate catalytic conversion rate,improved thermostability, activity at a different pH and/or optimizedcodon usage for improved expression in a host cell. The nucleic acidsequences can be preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higheridentity to the chimeric alkene synthase gene.

In one embodiment, the nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ IDNO: 20, or SEQ ID NO: 22. In another embodiment, the nucleic acidmolecule encodes a polypeptide having the amino acid sequence of SEQ IDNO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. Also provided isa nucleic acid molecule encoding a polypeptide sequence that is at least50% identical to either SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:31. Preferably, the nucleic acid molecule encodes a polypeptide sequenceof at least 55%, 60%, 70%, 80%, 90% or 95% identical to SEQ ID NO: 2,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:29, SEQ ID NO: 30, or SEQ ID NO: 31, and the identity can even morepreferably be 98%, 99%, 99.9% or even higher.

Provided also are nucleic acid molecules that hybridize under stringentconditions to the above-described nucleic acid molecules. As definedabove, and as is well known in the art, stringent hybridizations areperformed at about 25° C. below the thermal melting point (T_(m)) forthe specific DNA hybrid under a particular set of conditions, where theT_(m) is the temperature at which 50% of the target sequence hybridizesto a perfectly matched probe. Stringent washing can be performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions.

The nucleic acid molecule includes DNA molecules (e.g., linear,circular, cDNA, chromosomal DNA, double stranded or single stranded) andRNA molecules (e.g., tRNA, rRNA, mRNA) and analogs of the DNA or RNAmolecules of the described herein using nucleotide analogs. The isolatednucleic acid molecule of the invention includes a nucleic acid moleculefree of naturally flanking sequences (i.e., sequences located at the 5′and 3′ ends of the nucleic acid molecule) in the chromosomal DNA of theorganism from which the nucleic acid is derived. In various embodiments,an isolated nucleic acid molecule can contain less than about 10 kb, 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb, 50 bp, 25 bp or 10 bp ofnaturally flanking nucleotide chromosomal DNA sequences of themicroorganism from which the nucleic acid molecule is derived.

The chimeric alkene synthase genes, as described herein, include nucleicacid molecules, for example, a polypeptide or RNA-encoding nucleic acidmolecule, separated from another gene or other genes by intergenic DNA(for example, an intervening or spacer DNA which naturally flanks thegene and/or separates genes in the chromosomal DNA of the organism).

Nucleic acid molecules comprising a fragment of any one of theabove-described nucleic acid sequences are also provided. Thesefragments preferably contain at least 20 contiguous nucleotides. Morepreferably the fragments of the nucleic acid sequences contain at least25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguousnucleotides.

In another embodiment, an isolated alkene synthase-encoding nucleic acidmolecule hybridizes to all or a portion of a nucleic acid moleculehaving the nucleotide sequence set forth in SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; orhybridizes to all or a portion of a nucleic acid molecule having anucleotide sequence that encodes a polypeptide having the amino acidsequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31. The nucleic acid sequence fragmentsdisplay utility in a variety of systems and methods. For example, thefragments may be used as probes in various hybridization techniques.Depending on the method, the target nucleic acid sequences may be eitherDNA or RNA. The target nucleic acid sequences may be fractionated (e.g.,by gel electrophoresis) prior to the hybridization, or the hybridizationmay be performed on samples in situ. One of skill in the art willappreciate that nucleic acid probes of known sequence find utility indetermining chromosomal structure (e.g., by Southern blotting) and inmeasuring gene expression (e.g., by Northern blotting). In suchexperiments, the sequence fragments are preferably detectably labeled,so that their specific hybridization to target sequences can be detectedand optionally quantified. One of skill in the art will appreciate thatthe nucleic acid fragments may be used in a wide variety of blottingtechniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragmentsdisclosed herein also find utility as probes when immobilized onmicroarrays. Methods for creating microarrays by deposition and fixationof nucleic acids onto support substrates are well known in the art.Reviewed in DNA Microarrays: A Practical Approach (Practical ApproachSeries), Schena (ed.), Oxford University Press (1999) (ISBN:0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip:Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of which are incorporated herein by reference in theirentireties. Analysis of, for example, gene expression using microarrayscomprising nucleic acid sequence fragments, such as the nucleic acidsequence fragments disclosed herein, is a well-established utility forsequence fragments in the field of cell and molecular biology. Otheruses for sequence fragments immobilized on microarrays are described inGerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger,Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A PracticalApproach (Practical Approach Series), Schena (ed.), Oxford UniversityPress (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);Microarray Biochip: Tools and Technology, Schena (ed.), Eaton PublishingCompany/BioTechniques Books Division (2000) (ISBN: 1881299376), thedisclosures of each of which is incorporated herein by reference in itsentirety.

In another embodiment, the present disclosure provides isolated nucleicacid molecules encoding a chimeric alkene synthase which exhibitsincreased activity relative to the unmodified, native protein. Forexample, a particular chimeric alkene synthase may synthesize more1-pentadecene over a given time period, under identical conditions, whencompared to the unmodified native protein from which it is derived. Asis well known in the art, enzyme activities are measured in variousways, e.g. spectroscopically. (Grubmeyer et al., J. Biol. Chem.268:20299-20304 (1993)), or chromatographically, including the use ofhigh performance liquid chromatography (Chung and Sloan, J. Chromatogr.371:71-81 (1986)). As another alternative the activity is indirectlymeasured by determining the levels of product made from the enzymeactivity. More modern techniques include using gas chromatography linkedto mass spectrometry (Niessen, W. M. A. (2001). Current practice of gaschromatography—mass spectrometry. New York, N.Y: Marcel Dekker. (ISBN:0824704738)). Additional modern techniques for identification ofrecombinant protein activity and products including liquidchromatography-mass spectrometry (LCMS), high performance liquidchromatography (HPLC), capillary electrophoresis, Matrix-Assisted LaserDesorption Ionization time of flight-mass spectrometry (MALDI-TOF MS),nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy,viscometry (Knothe, G., R. O. Dunn, and M. O. Bagby. 1997. Biodiesel:The use of vegetable oils and their derivatives as alternative dieselfuels. Am. Chem. Soc. Symp. Series 666: 172-208), physicalproperty-based methods, wet chemical methods, etc. are used to analyzethe levels and the identity of the product produced by the organisms.Other methods and techniques may also be suitable for the measurement ofenzyme activity, as would be known by one of skill in the art.

Vectors

The recombinant vector can be altered, modified or engineered to havedifferent or a different quantity of nucleic acid sequences than in thederived or natural recombinant vector nucleic acid molecule. Preferably,the recombinant vector includes a gene or recombinant nucleic acidmolecule operably linked to regulatory sequences including, but notlimited to, promoter sequences, terminator sequences and/or artificialribosome binding sites (RBSs), as defined herein.

Typically, a gene encoding a chimeric alkene synthase is operably linkedto regulatory sequence(s) in a manner which allows for the desiredexpression characteristics of the nucleotide sequence. Preferably, thegene encoding a chimeric alkene synthase in a 1-nonadecene biosyntheticpathway is transcribed and translated into a gene product encoded by thenucleotide sequence when the recombinant nucleic acid molecule isincluded in a recombinant vector, as defined herein, and is introducedinto a microorganism.

The regulatory sequence may be comprised of nucleic acid sequences whichmodulate, regulate or otherwise affect expression of other nucleic acidsequences. In one embodiment, a regulatory sequence can be in a similaror identical position and/or orientation relative to a nucleic acidsequence as observed in its natural state, e.g., in a native positionand/or orientation. For example, a gene of interest can be included in arecombinant nucleic acid molecule or recombinant vector operably linkedto a regulatory sequence which accompanies or is adjacent to the gene ofinterest in the natural host cell, or can be adjacent to a differentgene in the natural host cell, or can be operably linked to a regulatorysequence from another organism. Regulatory sequences operably linked toa gene can be from other bacterial regulatory sequences, bacteriophageregulatory sequences and the like.

In one embodiment, a regulatory sequence is a sequence which has beenmodified, mutated, substituted, derivated, deleted, including sequenceswhich are chemically synthesized. Preferably, regulatory sequencesinclude promoters, enhancers, termination signals, anti-terminationsignals and other expression control elements that, for example, serveas sequences to which repressors or inducers bind or serve as or encodebinding sites for transcriptional and/or translational regulatorypolypeptides, for example, in the transcribed mRNA (see Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). Regulatory sequences includepromoters directing constitutive expression of a nucleotide sequence ina host cell, promoters directing inducible expression of a nucleotidesequence in a host cell and promoters which attenuate or repressexpression of a nucleotide sequence in a host cell. Regulatingexpression of a gene of interest also can be done by removing ordeleting regulatory sequences. For example, sequences involved in thenegative regulation of transcription can be removed such that expressionof a gene of interest is enhanced. In one embodiment, a recombinantnucleic acid molecule or recombinant vector includes a nucleic acidsequence or gene that encodes at least one chimeric alkene synthase inan alkene biosynthetic pathway, wherein the gene encoding the enzyme(s)is operably linked to a promoter or promoter sequence. Preferably,promoters include native promoters, surrogate promoters and/orbacteriophage promoters.

In one embodiment, a promoter is associated with a biochemicalhousekeeping gene or a promoter associated with an ethanologenicpathway. In another embodiment, a promoter is a bacteriophage promoter.Other promoters include tef (the translational elongation factor (TEF)promoter) which promotes high level expression in Bacillus (e.g.Bacillus subtilis). Additional advantageous promoters, for example, foruse in Gram positive microorganisms include, but are not limited to, theamyE promoter or phage SP02 promoters. Additional advantageouspromoters, for example, for use in Gram negative microorganisms include,but are not limited to tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq,T7, T5, T3, gal, trc, ara, SP6, λ-p_(R) or λ-p_(L).

In another embodiment, a recombinant nucleic acid molecule orrecombinant vector includes a transcription terminator sequence orsequences. Typically, terminator sequences refer to the regulatorysequences which serve to terminate transcription of a gene. Terminatorsequences (or tandem transcription terminators) can further serve tostabilize mRNA (e.g., by adding structure to mRNA), for example, againstnucleases.

In another embodiment, a recombinant nucleic acid molecule orrecombinant vector has sequences allowing for detection of the vectorcontaining sequences (i.e., detectable and/or selectable markers), forexample, sequences that overcome auxotrophic mutations, for example,ura3 or ilvE, fluorescent markers, and/or calorimetric markers (e.g.,lacZ/β-galactosidase), and/or antibiotic resistance genes (e.g., bla ortet).

It is understood that any one of the chimeric alkene synthase gene ofthe invention can be introduced into a vector also comprising one ormore genes involved in the biosynthesis of alkenes from light, water andinorganic carbon.

Also provided are vectors, including expression vectors, which comprisethe above nucleic acid molecules, as described further herein. In afirst embodiment, the vectors include the isolated nucleic acidmolecules described above. In an alternative embodiment, the vectorsinclude the above-described nucleic acid molecules operably linked toone or more expression control sequences. The vectors of the instantinvention may thus be used to express a polypeptide having chimericalkene synthase activity in an alkene biosynthetic pathway.

Vectors useful for expression of nucleic acids in prokaryotes are wellknown in the art. A useful vector herein is plasmid pCDF Duet-1 that isavailable from Novagen. Another useful vector is the endogenousSynechococcus sp. PCC 7002 plasmid pAQ1 (Genbank accession number NC010476).

Isolated Polypeptides

In one embodiment, polypeptides encoded by nucleic acid sequences areproduced by recombinant DNA techniques and can be isolated fromexpression host cells by an appropriate purification scheme usingstandard polypeptide purification techniques. In another embodiment,polypeptides encoded by nucleic acid sequences are synthesizedchemically using standard peptide synthesis techniques.

Included within the scope of the invention are chimeric alkene synthasepolypeptides or gene products that are derived polypeptides or geneproducts encoded by naturally-occurring bacterial genes. Further,included within the inventive scope, are bacteria-derived polypeptidesor gene products which differ from wild-type genes, including genes thathave altered, inserted or deleted nucleic acids but which encodepolypeptides substantially similar in structure and/or function to thewild-type and/or chimeric alkene synthase gene.

For example, it is well understood that one of skill in the art canmutate (e.g., substitute) nucleic acids which, due to the degeneracy ofthe genetic code, encode for an identical amino acid as that encoded bythe naturally-occurring gene. This may be desirable in order to improvethe codon usage of a nucleic acid to be expressed in a particularorganism. Moreover, it is well understood that one of skill in the artcan mutate (e.g., substitute) nucleic acids which encode forconservative amino acid substitutions. It is further well understoodthat one of skill in the art can substitute, add or delete amino acidsto a certain degree to improve upon or at least insubstantially affectthe function and/or structure of a gene product (e.g., alcoholdehydrogenase activity) as compared with a naturally-occurring geneproduct, each instance of which is intended to be included within thescope of the invention.

In various aspects, isolated polypeptides (including muteins, allelicvariants, fragments, derivatives, and analogs) encoded by the nucleicacid molecules are provided. In one embodiment, the isolated polypeptidecomprises the polypeptide sequence corresponding to SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:31. In an alternative embodiment, the isolated polypeptide comprises apolypeptide sequence at least 50% identical to SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31.Preferably the isolated polypeptide has preferably 50%, 60%-70%,70%-80%, 80%-90%, 90%-95%, 95%-98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%,98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or even higher identity to the sequencesoptimized for substrate affinity and/or substrate catalytic conversionrate.

According to other embodiments, isolated polypeptides comprising afragment of the above-described polypeptide sequences are provided.These fragments preferably include at least 20 contiguous amino acids,more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 oreven more contiguous amino acids.

The polypeptides also include fusions between the above-describedpolypeptide sequences and heterologous polypeptides. The heterologoussequences can, for example, include sequences designed to facilitatepurification, e.g. histidine tags, and/or visualization ofrecombinantly-expressed proteins. Other non-limiting examples of proteinfusions include those that permit display of the encoded protein on thesurface of a phage or a cell, fusions to intrinsically fluorescentproteins, such as green fluorescent protein (GFP), and fusions to theIgG Fc region.

Host Cell Transformants

In other aspects, host cells transformed with the nucleic acid moleculesor vectors, and descendants thereof, are provided. In some embodiments,these cells carry the nucleic acid sequences on vectors, which may butneed not be freely replicating vectors. In other embodiments, thenucleic acids have been integrated into the genome of the host cells.

In a preferred embodiment, the host cell comprises one or more nucleicacids of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 26, SEQID NO: 27, or SEQ ID NO: 28 operably linked to promoters for theexpression of chimeric alkene synthase in an alkene biosynthesispathway.

In another embodiment, the host cell containing a chimeric alkenesynthase in the alkene pathway is suitable for producing 1-alkenes. In aparticular embodiment, the host cell is a recombinant host cell thatproduces 1-alkenes comprising a chimeric nucleic acid encoding a nucleicacid of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 26, SEQID NO: 27, or SEQ ID NO: 28.

In certain aspects, methods for expressing a polypeptide under suitableculture conditions and choice of host cell line for optimal enzymeexpression, activity and stability (codon usage, salinity, pH,temperature, etc.) are provided.

In another aspect, methods for producing 1-alkene by culturing a hostcell under conditions in which the chimeric alkene synthase is expressedat sufficient levels to produce a measureable quantity of 1-alkene aredescribed. In a related embodiment, methods for producing 1-alkene areperformed by contacting a cell lysate obtained from the above host cellunder conditions in which 1-alkene is produced from light, water andinorganic carbon. Accordingly, the present disclosure provides enzymeextracts having chain-length specific alkene synthase activity, andhaving, for example, thermal stability, activity at various pH, and/orsuperior substrate affinity or specificity.

Selected or Engineered Microorganisms for the Production of Carbon-BasedProducts of Interest

Microorganism: Includes prokaryotic and eukaryotic microbial speciesfrom the Domains Archaea, Bacteria and Eucarya, the latter includingyeast and filamentous fungi, protozoa, algae, or higher Protista. Theterms “microbial cells” and “microbes” are used interchangeably with theterm microorganism.

A variety of host organisms can be transformed to produce a product ofinterest. Photoautotrophic organisms include eukaryotic plants andalgae, as well as prokaryotic cyanobacteria, green-sulfur bacteria,green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfurbacteria.

Host cells can be a Gram-negative bacterial cell or a Gram-positivebacterial cell. A Gram-negative host cell of the invention can be, e.g.,Gluconobacter, Rhizobium, Bradyrhizobium, Alcaligenes, Rhodobacter,Rhodococcus. Azospirillum, Rhodospirillum, Sphingomonas, Burkholderia,Desulfomonas, Geospirillum, Succinomonas, Aeromonas, Shewanella,Halochromatium, Citrobacter, Escherichia, Klebsiella, ZymomonasZymobacter, or Acetobacter. A Gram-positive host cell of the inventioncan be, e.g., Fibrobacter, Acidobacter, Bacteroides, Sphingobacterium,Actinomyces, Corynebacterium, Nocardia, Rhodococcus, Propionibacterium,Bifidobacterium, Bacillus, Geobacillus, Paenibacillus, Sulfobacillus,Clostridium, Anaerobacter, Eubacterium, Streptococcus, Lactobacillus,Leuconostoc, Enterococcus, Lactococcus, Thermobifida, Cellulomonas, orSarcina.

Extremophiles are also contemplated as suitable organisms. Suchorganisms withstand various environmental parameters such astemperature, radiation, pressure, gravity, vacuum, desiccation,salinity, pH, oxygen tension, and chemicals. They includehyperthermophiles, which grow at or above 80° C. such as Pyrolobusfumarii; thermophiles, which grow between 60-80° C. such asSynechococcus lividis; mesophiles, which grow between 15-60° C. andpsychrophiles, which grow at or below 15° C. such as Psychrobacter andsome insects. Radiation tolerant organisms include Deinococcusradiodurans. Pressure tolerant organisms include piezophiles orbarophiles which tolerate pressure of 130 MPa. Hypergravity (e.g., >1 g)hypogravity (e.g., <1 g) tolerant organisms are also contemplated.Vacuum tolerant organisms include tardigrades, insects, microbes andseeds. Dessicant tolerant and anhydrobiotic organisms include xerophilessuch as Artemia salina; nematodes, microbes, fungi and lichens. Salttolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriaceaand Dunaliella salina. pH tolerant organisms include alkaliphiles suchas Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH>9)and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., lowpH). Anaerobes, which cannot tolerate O₂ such as Methanococcusjannaschii; microaerophils, which tolerate some O₂ such as Clostridiumand aerobes, which require O₂ are also contemplated. Gas tolerantorganisms, which tolerate pure CO₂ include Cyanidium caldarium and metaltolerant organisms include metalotolerants such as Ferroplasmaacidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH₃₄ (e.g., Zn, Co,Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing CreaturesThriving in Extreme Environments. New York: Plenum (1998) and Seckbach,J. “Search for Life in the Universe with Terrestrial Microbes WhichThrive Under Extreme Conditions.” In Cristiano Batalli Cosmovici, StuartBowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Originsand the Search for Life in the Universe, p. 511. Milan: EditriceCompositori (1997).

Plants include but are not limited to the following genera: Arabidopsis,Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus,Saccharum, Salix, Simmondsia and Zea.

Algae and cyanobacteria include but are not limited to the followinggenera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes,Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium,Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora,Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus,Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa,Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus,Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella,Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys,Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis,Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira,Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis,Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena,Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros,Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis,Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris,Characiopsis, Characium, Charales, Chilomonas, Chlainomonas,Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis,Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella,Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea,Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema,Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton,Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas,Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa,Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus,Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta,Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera,Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium,Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis,Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella,Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema,Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis,Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis,Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta,Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia,Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta,Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis,Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax,Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia,Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga,Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea,Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium,Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia,Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema,Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium,Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne,Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron,Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium,Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia,Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion,Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis,Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella,Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira,Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias,Microchaete, Microcoleus, Microcystis, Microglena, Micromonas,Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus,Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris,Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium,Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema,Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria,Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus,Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas,Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium,Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium,Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis,Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora,Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema,Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus,Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra,Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis,Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella,Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus,Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma,Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium,Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis,Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas,Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma,Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus,Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella,Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium,Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella,Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix,Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria,Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella,Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria,Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.

Green non-sulfur bacteria include but are not limited to the followinggenera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix,Herpetosiphon, Roseiflexus, and Thermomicrobium.

Green sulfur bacteria include but are not limited to the followinggenera: Chlorobium, Clathrochloris, and Prosthecochloris.

Purple sulfur bacteria include but are not limited to the followinggenera: Allochromatium, Chromatium, Halochromatium, Isochromatium,Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa,Thiorhodococcus, and Thiocystis,

Purple non-sulfur bacteria include but are not limited to the followinggenera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium,Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum,Rodovibrio, and Roseospira.

Aerobic chemolithotrophic bacteria include but are not limited tonitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp.,Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp.,Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibriosp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligatelychemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., ironand manganese-oxidizing and/or depositing bacteria such as Siderococcussp., and magnetotactic bacteria such as Aquaspirillum sp.

Archaeobacteria include but are not limited to methanogenicarchaeobacteria such as Methanobacterium sp., Methanobrevibacter sp.,Methanothermus sp., Methanococcus sp., Methanomicrobium sp.,Methanospirillum sp., Methanogenium sp., Methanosarcina sp.,Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanussp.; extremely thermophilic sulfur-metabolizers such as Thermoproteussp., Pyrodictium sp., Sulfolobus sp., Acidianus sp. and othermicroorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae,Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp.,Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.

HyperPhotosynthetic conversion requires extensive genetic modification;thus, in preferred embodiments the parental photoautotrophic organismcan be transformed with exogenous DNA.

Preferred organisms for HyperPhotosynthetic conversion include:Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zeamays (plants), Botryococcus braunii, Chlamydomonas reinhardtii andDunaliela salina (algae), Synechococcus sp PCC 7002, Synechococcus sp.PCC 7942, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatusBP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria),Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidumand Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum,Rhodobacter capsulatus, and Rhodopseudomonas palusris (purple non-sulfurbacteria).

Yet other suitable organisms include synthetic cells or cells producedby synthetic genomes as described in Venter et al. US Pat. Pub. No.2007/0264688, and cell-like systems or synthetic cells as described inGlass et al. US Pat. Pub. No. 2007/0269862.

Still, other suitable organisms include microorganisms that can beengineered to fix inorganic carbon, such as Escherichia coli,Acetobacter aceti, Bacillus subtilis, yeast and fungi such asClostridium ljungdahlii, Clostridium thermocellum, Penicilliumchrysogenum, Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonasmobilis.

A common theme in selecting or engineering a suitable organism isautotrophic fixation of CO₂ to products. This would cover photosynthesisand methanogenesis. Acetogenesis, encompassing the three types of CO₂fixation; Calvin cycle, acetyl CoA pathway and reductive TCA pathway isalso covered. The capability to use carbon dioxide as the sole source ofcell carbon (autotrophy) is found in almost all major groupsofprokaryotes. The CO₂ fixation pathways differ between groups, andthere is no clear distribution pattern of the four presently-knownautotrophic pathways. Fuchs, G. 1989. Alternative pathways ofautotrophic CO₂ fixation, p. 365-382. In H. G. Schlegel, and B. Bowien(ed.), Autotrophic bacteria. Springer-Verlag, Berlin, Germany. Thereductive pentose phosphate cycle (Calvin-Bassham-Benson cycle)represents the CO₂ fixation pathway in many aerobic autotrophicbacteria, for example, cyanobacteria.

Gene Integration and Propagation

The 1-alkene producing genes can be propagated by insertion into thehost cell genome. Integration into the genome of the host cell isoptionally done at particular loci to impair or disable unwanted geneproducts or metabolic pathways.

In another embodiment is described the integration of a chimeric alkenesynthase gene into a plasmid. The plasmid can express one or more genes,optionally an operon including one or more genes, preferably one or morechimeric genes involved in the synthesis of 1-alkene, or more preferablyone or more chimeric genes of a related metabolic pathway that feedsinto the biosynthetic pathway for 1-alkenes.

Antibodies

In another aspect, provided herein are isolated antibodies, includingfragments and derivatives thereof that bind specifically to the isolatedpolypeptides and polypeptide fragments or to one or more of thepolypeptides encoded by the isolated nucleic acids. The antibodies maybe specific for linear epitopes, discontinuous epitopes orconformational epitopes of such polypeptides or polypeptide fragments,either as present on the polypeptide in its native conformation or, insome cases, as present on the polypeptides as denatured, as, e.g., bysolubilization in SDS. Among the useful antibody fragments are Fab,Fab′, Fv, F(ab′)₂, and single chain Fv fragments.

By “bind specifically” and “specific binding” is here intended theability of the antibody to bind to a first molecular species inpreference to binding to other molecular species with which the antibodyand first molecular species are admixed. An antibody is saidspecifically to “recognize” a first molecular species when it can bindspecifically to that first molecular species.

As is well known in the art, the degree to which an antibody candiscriminate as among molecular species in a mixture will depend, inpart, upon the conformational relatedness of the species in the mixture;typically, the antibodies will discriminate over adventitious binding tounrelated polypeptides by at least two-fold, more typically by at least5-fold, typically by more than 10-fold, 25-fold, 50-fold, 75-fold, andoften by more than 100-fold, and on occasion by more than 500-fold or1000-fold.

Typically, the affinity or avidity of an antibody (or antibody multimer,as in the case of an IgM pentamer) for a polypeptide or polypeptidefragment will be at least about 1×10⁻⁶ M, typically at least about5×10⁻⁷ M, usefully at least about 1×10⁻⁷ M, with affinities andavidities of 1×10⁻⁸ M, 5×10⁻⁹ M, 1×10⁻¹⁰ M and even stronger provingespecially useful.

The isolated antibodies may be naturally-occurring forms, such as IgG,IgM, IgD, IgE, and IgA, from any mammalian species. For example,antibodies are usefully obtained from species includingrodents-typically mouse, but also rat, guinea pig, andhamster-lagomorphs, typically rabbits, and also larger mammals, such assheep, goats, cows, and horses. The animal is typically affirmativelyimmunized, according to standard immunization protocols, with thepolypeptide or polypeptide fragment.

Virtually all fragments of 8 or more contiguous amino acids of thepolypeptides may be used effectively as immunogens when conjugated to acarrier, typically a protein such as bovine thyroglobulin, keyholelimpet hemocyanin, or bovine serum albumin, conveniently using abifunctional linker. Immunogenicity may also be conferred by fusion ofthe polypeptide and polypeptide fragments to other moieties. Forexample, peptides can be produced by solid phase synthesis on a branchedpolylysine core matrix; these multiple antigenic peptides (MAPs) providehigh purity, increased avidity, accurate chemical definition andimproved safety in vaccine development. See, e.g., Tam et al., Proc.Natl. Acad. Sci. USA 85:5409-5413 (1988); Posnett et al., J. Biol. Chem.263, 1719-1725 (1988).

Protocols for immunization are well-established in the art. Suchprotocols often include multiple immunizations, either with or withoutadjuvants such as Freund's complete adjuvant and Freund's incompleteadjuvant. Antibodies may be polyclonal or monoclonal, with polyclonalantibodies having certain advantages in immunohistochemical detection ofthe proteins and monoclonal antibodies having advantages in identifyingand distinguishing particular epitopes of the proteins. Followingimmunization, the antibodies may be produced using any art-acceptedtechnique. Host cells for recombinant antibody production—either wholeantibodies, antibody fragments, or antibody derivatives—can beprokaryotic or eukaryotic. Prokaryotic hosts are particularly useful forproducing phage displayed antibodies, as is well known in the art.Eukaryotic cells, including mammalian, insect, plant and fungal cellsare also useful for expression of the antibodies, antibody fragments,and antibody derivatives. Antibodies can also be prepared by cell freetranslation.

The isolated antibodies, including fragments and derivatives thereof,can usefully be labeled. It is, therefore, another aspect to providelabeled antibodies that bind specifically to one or more of thepolypeptides and polypeptide fragments. The choice of label depends, inpart, upon the desired use. In some cases, the antibodies may usefullybe labeled with an enzyme. Alternatively, the antibodies may be labeledwith colloidal gold or with a fluorophore. For secondary detection usinglabeled avidin, streptavidin, captavidin or neutravidin, the antibodiesmay usefully be labeled with biotin. When the antibodies are used, e.g.,for Western blotting applications, they may usefully be labeled withradioisotopes, such as ³³P, ³²P, ³⁵S, ³H and ¹²⁵I. As would beunderstood, use of the labels described above is not restricted to anyparticular application.

Methods for Designing Chimeric Protein Variants

Chain length-specific alkene production can be achieved through theexpression and optimization of chimeric alkene synthase in organismswell suited for modern genetic engineering techniques, i.e., those thatrapidly grow, are capable of thriving on inexpensive food resources andfrom which isolation of a desired product is easily and inexpensivelyachieved. To control the chain length of alkene production it would beadvantageous to design and select variants of the chimeric enzymes,including but not limited to, variants optimized for substrate affinity,substrate specificity, substrate catalytic conversion rate, improvedthermostability, activity at a different pH and/or optimized codon usagefor improved expression in a host cell. See, for example, amino acidchanges correlated to alterations in the catalytic rate whilemaintaining similar affinities (R L Zheng and R G Kemp, J. Biol. Chem.(1994) Vol. 269:18475-18479) or amino acid changes correlated withchanges in the stability of the transition state that affect catalyticturnover (MA Phillips, et al., J. Biol. Chem., (1990) Vol.265:20692-20698). It would be another advantage to design and select forchimeric enzymes altered to have substantially decreased reversereaction activity in which enzyme-substrate products would be the resultof energetically unfavorable bond formation or molecularre-configuration of the substrate, and have improved forward reactionactivity in which enzyme-substrate products would be the result ofenergetically favorable molecular bond reduction or molecularre-configuration.

Accordingly, one method for the design of improved chimeric alkenesynthase proteins for synthesing 1-alkenes utilizes computational andbioinformatic analysis to design and select for advantageous changes inchimeric amino acid sequences encoding alkene synthase enzyme activity.Computational methods and bioinformatics provide tractable alternativesfor rational design of protein structure and function. Recently,algorithms analyzing protein structure for biophysical character (forexample, motional dynamics and total energy or Gibb's Free Energyevaluations) have become a commercially feasible methodologysupplementing protein sequence analysis data that assess homology,identity and/or degree of sequence and domain conservation to improveupon or design the desirable qualities of a protein (Rosetta++,University of Washington). For example, an in silico redesign of theendonuclease I-MsoI was based on computational evaluation of biophysicalparameters of rationally selected changes to the primary amino acidsequence. Researchers were able to maintain wild-type bindingselectivity and affinity yet improve the catalytic turnover by fourorders of magnitude (Ashworth, et al., Nature (2006) vol. 441:656-659).

In one embodiment, chimeric polypeptide sequences or related homologuesin a complex with a substrate are obtained for computational analysis onsteady state and/or changes in Gibb's free energy relative to the wildtype protein. Substitutions of one amino acid residue for another areaccomplished in silico interactively as a means for identifying specificresidue substitutions that optimize structural or catalytic contactsbetween the protein and substrate using standard software programs forviewing molecules as is well known to those skilled in the art. To theextent that in silico structures for the chimeric polypeptides (andhomologues) described herein are available, those structures can be usedto rationally design modified proteins with desired (typically,improved) activities. Specific amino acid substitutions are rationallychosen based on substituted residue characteristics that optimize, forexample, Van der Waal's interactions, hydrophobicity, hydrophilicity,steric non-interferences, pH-dependent electrostatics and relatedchemical interactions. The overall energetic change of the substitutionprotein model when unbound and bound to its substrate is calculated andassessed by one having skill in the art to be evaluated for the changein free energy for correlations to overall structural stability (e.g.,Meiler, J. and D. Baker, Proteins (2006) 65:538-548). In addition, suchcomputational methods provide a means for accurately predictingquaternary protein structure interactions such that in silicomodifications are predictive or determinative of overall multimericstructural stability (Wollacott, A M, et al., Protein Science (2007)16:165-175; Joachimiak, L A, et al., J. Mol. Biol. (2006) 361:195-208).

Preferably, a rational design change to the primary structure ofchimeric alkene synthase protein sequences minimally alter the Gibb'sfree energy state of the unbound polypeptide and maintain a folded,functional and similar wild-type enzyme structure. More preferably alower computational total free energy change of the protein sequence isachieved to indicate the potential for optimized enzyme structuralstability.

Although lower free energy of a protein structure relative to theoriginal chimeric structure is an indicator of thermodynamic stability,the positive correlation of increased thermal stability to optimizedfunction does not always exist. Therefore, preferably, optimal catalyticcontacts between the modified chimeric alkene synthase and the substrateare achieved with a concomitant predicted favorable change in total freeenergy of the catabolic reaction, for example by rationally designingchimeric alkene synthase protein/substrate interactions that stabilizethe transition state of the enzymatic reaction while maintaining asimilar or favorable change in free energy of the unbound chimericalkene synthase protein for a desired environment in which a host cellexpresses the mutant chimeric alkene synthase protein. Even morepreferably, rationally selected amino acid changes result in asubstantially decreased chimeric alkene synthase enzyme's anabolicprotein/substrate reaction or increase the chimeric alkene synthasesprotein/substrate reaction, for example wherein specific chain-length1-alkenes are synthesized for a desired environment in which a host cellexpresses the mutant chimeric alkene synthase. In a further embodimentany and/or all chimeric alkene synthase sequences are expressionoptimized for the specific expression host cell.

Methods for Generating Protein Variants

Several methods well known to those with skill in the art are availableto generate random nucleotide sequence variants for a correspondingchimeric polypeptide sequence using the Polymerase Chain Reaction(“PCR”) (U.S. Pat. No. 4,683,202). One embodiment is the generation ofchimeric alkene synthase gene variants using the method of error pronePCR. (R. Cadwell and G. Joyce, PCR Meth. Appl. (1991) Vol. 2:28-33;Leung, et al., Technique (1989) Vol. 1:11-15). Error prone PCR isachieved by the establishment of a chemical environment during the PCRexperiment that causes an increase in unfaithful replication of a parentcopy of DNA sought to be replicated. For example, increasing themanganese or magnesium ion content of the chemical admixture used in thePCR experiment, very low annealing temperatures, varying the balanceamong di-deoxy nucleotides added, starting with a low population ofparent DNA templates or using polymerases designed to have increasedinefficiencies in accurate DNA replication all result in nucleotidechanges in progeny DNA sequences during the PCR replication process. Theresultant mutant DNA sequences are genetically engineered into anappropriate vector to be expressed in a host cell and analyzed to screenand select for the desired effect on whole cell production of a productor process of interest. In one embodiment, random mutagenesis of thechimeric alkene synthase-encoding nucleotide sequences is generatedthrough error prone PCR using techniques well known to one skilled inthe art. Resultant nucleotide sequences are analyzed for structural andfunctional attributes through clonal screening assays and other methodsas described herein.

Another embodiment is generating a specifically desired protein mutantusing site-directed mutagenesis. For example, with overlap extension(An, et al., Appl. Microbiol. Biotech. (2005) vol. 68(6):774-778) ormega-primer PCR (E. Burke and S. Batik, Methods Mol. Bio. (2003) vol226:525-532) one can use nucleotide primers that have been altered atcorresponding codon positions in the parent nucleotide to yield DNAprogeny sequences containing the desired mutation. Alternatively, onecan use cassette mutagenesis (Kegler-Ebo, et al., Nucleic Acids Res.(1994) vol. 22(9):1593-1599) as is commonly known by one skilled in theart.

Several authors (Korkhin, et al., J. Mol. Bio. (1998) vol. 278:967-981;E. Goiberg, et al., Proteins (2008) vol. 72:711-719) have demonstratedprotein amino acid substitutions at single positions in the alcoholdehydrogenase protein sequence enhance protein fold thermostability. Inone aspect, using site-directed mutagenesis and cassette mutagenesis,all possible positions in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31 are changed to a proline,transformed into a suitable high expression vector and expressed at highlevels in a suitable expression host cell. Purified aliquots atconcentrations necessary for the appropriate biophysical analyticaltechnique are obtained by methods as known to those with skill in theart (P. Rellos and R. K. Scopes, Prot. Exp. Purific. (1994) Vol.5:270-277) and evaluated for increased thermostability.

Another embodiment is to select for a polypeptide variant for expressionin a recipient host cell by comparing a first nucleic acid sequenceencoding the polypeptide with the nucleic acid sequence of a second,related nucleic acid sequence encoding a polypeptide having moredesirable qualities, and altering at least one codon of the firstnucleic acid sequence to have identity with the corresponding codon ofthe second nucleic acid sequence, such that improved polypeptideactivity, substrate specificity, substrate affinity (for example, NADPHand acetaldehyde), substrate catalytic conversion rate, improvedthermostability, activity at a different pH and/or optimized codon usagefor expression and/or structure of the altered polypeptide is achievedin the host cell.

In yet another embodiment, all amino acid residue variations are encodedat any desired, specified nucleotide codon position using such methodsas site saturation mutagenesis (Meyers, et al., Science (1985) Vol.229:242-247; Derbyshire, et al., Gene (1986) Vol. 46:145-152; U.S. Pat.No. 6,171,820). Whole gene site saturation mutagenesis (K. Kretz, etal., Meth. Enzym. (2004) Vol. 388:3-11) is preferred wherein all aminoacid residue variations are encoded at every nucleotide codon position.Both methods yield a population of protein variants differing from theparent polypeptide by one amino acid, with each amino acid substitutionbeing correlated to structural/functional attributes at any position inthe polypeptide. Saturation mutagenesis uses PCR and primers homologousto the parent sequence wherein one or more codon encoding nucleotidetriplets is randomized. Randomization results in the incorporation ofcodons corresponding to all amino acid replacements in the final,translated polypeptide. Each PCR product is genetically engineered intoan expression vector to be introduced into an expression host andscreened for structural and functional attributes through clonalscreening assays and other methods as described herein.

In one aspect of saturation mutagenesis, correlated saturationmutagenesis (“CSM”) is used wherein two or more amino acids atrationally designated positions are changed concomitantly to differentamino acid residues to engineer improved enzyme function and structure.Correlated saturation mutagenesis allows for the identification ofcomplimentary amino acid changes having positive, synergistic effects onchimeric alkene synthase enzyme structure and function. Such synergisticeffects include, but are not limited to, significantly altered enzymestability, substrate affinity, substrate specificity or catalyticturnover rate, independently or concomitantly increasing advantageouslythe production of 1-alkenes.

In yet another embodiment, amino acid substitution combinations of CSMderived protein variants being optimized for a particular function arecombined with one or more CSM derived protein variants being optimizedfor another particular function to derive a chimeric alkene synthaseprotein variant exhibiting multiple optimized structural and functionalcharacteristics. For example, amino acid changes in combinatorialmutants showing optimized protomer interactions are combined with aminoacid changes in combinatorial mutants showing optimized catalyticturnover.

In one embodiment, mutational variants derived from the methodsdescribed herein are cloned. DNA sequences produced by saturationmutagenesis are designed to have restriction sites at the ends of thegene sequences to allow for excision and transformation into a host cellplasmid. Generated plasmid stocks are transformed into a host cell andincubated at optimal growth conditions to identify successfullytransformed colonies.

In a further embodiment any and/or all sequences additionally areexpression optimized for the specific expression host cell.

Methods for Measuring Protein Variant Efficacy

Variations in expressed polypeptide sequences may result in measurabledifferences in the whole-cell rate of substrate conversion. It isdesirable to determine differences in the rate of substrate conversionby assessing productivity in a host cell having a particular proteinvariant relative to other whole cells having a different proteinvariant. Additionally, it would be desirable to determine the efficaciesof whole-cell substrate conversion as a function of environmentalfactors including, but not limited to, pH, temperature nutrientconcentration and salinity.

Therefore, in one embodiment, the biophysical analyses described hereinon protein variants are performed to measure structural/functionalattributes. Standard analyses of polypeptide activity are well known toone of ordinary skill in the art. Such analysis can require theexpression and high purification of large quantities of polypeptide,followed by various physical methods (including, but not limited to,calorimetry, fluorescence, spectrophotometric, spectrometric, liquidchromatography (LC), mass spectrometry (MS), LC-MS, affinitychromatography, light scattering, nuclear magnetic resonance and thelike) to assay function in a specific environment or functionaldifferences among homologues.

In another embodiment, the polypeptides are expressed, purified andsubject to the aforementioned analytical techniques to assess thefunctional difference among polypeptide sequence homologues, forexample, the rate of substrate conversion specific for a particularenzyme function.

Batch culture (or closed system culture) analysis is well known in theart and can provide information on host cell population effects for hostcells expressing genetically engineered genes. In batch cultures a hostcell population will grow until available nutrients are depleted fromthe culture media.

In one embodiment, the polypeptides are expressed in a batch culture andanalyzed for approximate doubling times, expression efficacy of theengineered polypeptide and end-point net product formation and netbiomass production.

Turbidostats are well known in the art as one form of a continuousculture within which media and nutrients are provided on anuninterrupted basis and allow for non-stop propagation of host cellpopulations. Turbidostats allow the user to determine information onwhole cell propagation and steady-state productivity for a particularbiologically produced end product such as host cell doubling time,temporally delimited biomass production rates for a particular host cellpopulation density, temporally delimited host cell population densityeffects on substrate conversion and net productivity of a host cellsubstrate conversion of, for example, octadecanoic acid to 1-nonadecene.Turbidostats can be designed to monitor the partitioning of substrateconversion products to the liquid or gaseous state. Additionally,quantitative evaluation of net productivity of a carbon-based product ofinterest can be accurately performed due to the exacting level ofcontrol that one skilled in the art has over the operation of theturbidostat. These types of information are useful to assess the parsedand net efficacies of a host cell genetically engineered to produce aspecific carbon-based product of interest.

In one embodiment, identical host cell lines differing only in thenucleic acid and expressed polypeptide sequence of a homologous enzymeare cultured in a uniform-environment turbidostat to determine highestwhole cell efficacy for the desired carbon-based product of interest.

In another embodiment, identical host cell lines differing only in thenucleic acid and expressed polypeptide sequence of a homologous enzymeare cultured in a batch culture or a turbidostat in varying environments(e.g. temperature, pH, salinity, nutrient exposure) to determine highestwhole cell efficacy for the desired carbon-based product of interest.

In one embodiment, mutational variants derived from the methodsdescribed herein are cloned. DNA sequences produced by saturationmutagenesis are designed to have restriction sites at the ends of thegene sequences to allow for cleavage and transformation into a host cellplasmid. Generated plasmid stocks are transformed into a host cell andincubated at optimal growth conditions to identify successfullytransformed colonies.

In one embodiment, to select protein variants, a colorimetric assay isused to screen for acetaldehyde to qualitatively determine the activityof variants of chimeric alkene synthase in a 1-alkene biosyntheticpathway.

Methods for Producing 1-Alkenes

It is desirable to engineer into an organism suited for industrial use agenetic system from which a chain length-specific 1-alkene can beproduced efficiently and cleanly.

Accordingly, the invention includes the conversion of water, inorganiccarbon, and light into a selected 1-alkene using the chimeric alkenesynthase described herein. In one embodiment, the genetically engineeredhost cells expresses a chimeric alkene synthase and one or more genes inan alkene biosynthetic pathway enabling the host cell to convert water,light and inorganic carbon and/or a selected 1-alkene precursor into aspecific pre-selected 1-alkene.

In another embodiment of the invention, the genetically engineered hostcell is processed into an enzymatic lysate for performing the aboveconversion reaction. In yet another embodiment, the chimeric alkenesynthase is purified, as described herein, for carrying out theconversion reaction.

The host cells and/or enzymes, for example in the lysate, partiallypurified, or purified, used in the conversion reactions are in a formallowing them to perform their intended function, producing a desired1-alkene, for example, 1-pentadecene. The microorganisms used can bewhole cells, or can be only those portions of the cells necessary toobtain the desired end result. The microorganisms can be suspended(e.g., in an appropriate solution such as buffered solutions or media),rinsed (e.g., rinsed free of media from culturing the microorganism),acetone-dried, immobilized (e.g., with polyacrylamide gel ork-carrageenan or on synthetic supports, for example, beads, matrices andthe like), fixed, cross-linked or permeabilized (e.g., havepermeabilized membranes and/or walls such that compounds, for example,substrates, intermediates or products can more easily pass through saidmembrane or wall).

In yet another embodiment, purified or unpurified chimeric alkenesynthase enzymes (e.g., SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31) are used in the conversionreactions. The enzyme is in a form that allows it to perform itsintended function. For example, the enzyme can be immobilized,conjugated or floating freely.

The following examples are for illustrative purposes and are notintended to limit the scope of the invention.

Example 1 Identification and Characterization of the Acyl Binding Pocketof Alkene Synthase

NonA has several catalytic domains (FIG. 2) with a LuxE-superfamilyacyltransferase domain at the N-terminus. This domain serves to load aC18, C17 or C16 acyl chain to the acyl-carrier protein (ACP) domain(i.e. the acyl binding pocket or the interior acyl binding pocket)triggering the biosynthetic pathway of 1-alkenes (FIG. 3, showing1-nonadecene biosynthesis from a C18:0 acyl chain substrate). In orderto identify the acyl binding pocket of NonA, the primary amino acidsequence was aligned with the acyl binding pocket of saframycin M×1synthetase B (i.e. SafB) (Li et al. 2008) for which two crystalstructures exist with the protein in a complex with5′-O-[(S)(dodecanoyloxy)(hydroxy) phosphoryl]adenosine (PDB #3KXW,3LNV). The amino acids comprising the acyl binding pocket were annotatedusing PyMOL 0.99rc6 by identifying the amino acids located fiveangstroms or less from the acyl-adenylate ligand (FIG. 4A).

Alignment of the amino acids comprising the SafB acyl binding pocketdomain with the corresponding amino acids in NonA and two other acylbinding pocket domains of known substrate specificity for saturated acylchains of different lengths (Table 1) showed that each acyl bindingpocket domain is strongly conserved towards the front of the acylbinding pocket (FIG. 4B, FIG. 5). One residue (327) of SafB at the frontof the acyl binding pocket was changed from leucine in SafB tomethionine in NonA (FIG. 4B, FIG. 5). This residue may play a role withsubstrate selectivity, as the other enzymes specifically bind acyl-CoAor acyl-adenylate substrates. The amino acid Ser374, which was close tothe adenylate core, is not conserved in all four enzymes and isseparated from Cys324 by 4.2 angstroms. Cys324 is at the front of theacyl binding pocket and is also not conserved (FIG. 4B). Ser374-Cys324(also found in NonA) therefore may be important in stabilizing thepocket. The amino acid residues toward the back of the pocket variedconsiderably between the four enzymes (FIG. 4B, FIG. 5) as would beexpected given their anticipated role in chain length selectivity.

Example 2 Identification of NonA Synthase Enzymes with Varied AlkeneSubstrate Specificity

Two conserved regions in the primary amino acid sequence of the fouracyl binding pocket domains were identified that flanked the interioracyl binding pocket (IABP) of the SafB acyl binding pocket domain. Theseconserved regions were aligned with NonA as described above and used asthe points to designate where to replace the IABP sequence of NonA withIABP sequences of the three other heterologous enzymes, each having aunique chain length specificity (FIG. 5). The IABP residues in SafB are197-294 (SEQ ID NO:8), and spatially these amino acids form a compactsubunit in SafB (FIG. 6A). The IABP residues surround the middle to endof the acyl chain of the ligand (FIG. 6B) and comprise the surroundingpocket.

Example 3 Engineering and Expression of Chimeric NonA Synthase

The corresponding nucleotides of the acyl binding pocket of NonA (SEQ IDNO: 3) are replaced with the coding nucleotides for the acyl bindingpocket of SafB (SEQ ID NO:7), MycA (SEQ ID NO:11), or DptE (SEQ IDNO:15), resulting in a NonA chimeric alkene synthase encoded by SEQ IDNO:17, SEQ ID NO:19, or SEQ ID NO:21. The chimeric NonA alkene synthaseenzyme comprises a heterologous acyl binding pocket with its native NonAIABP amino acid residues (SEQ ID NO:4) replaced with the IABP aminoacids from SafB (SEQ ID NO: 8), MycA (SEQ ID NO:12), and DptE (SEQ IDNO:16). The resulting chimeric alkene synthase has a polypeptidesequence of SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

The resulting chimeric alkene synthases are assayed and characterized bytheir differing acyl-binding pocket specificities. The pre-determinedspecific chain length 1-alkenes produced by a chimeric NonA alkenesynthase having a heterologous acyl binding pocket are consistent withthe chain length specificities of the protein source of the acyl bindingpocket as shown in Table 1, where the last column indicates the expected1-alkenes produced by a chimeric NonA alkene synthase containing aheterologous interior acyl binding pocket from the indicated proteins.

TABLE 1 Proteins that contain acyl binding pockets and their anticipatedsubstrate preference for fatty acids. Chain length Expected 1- ProteinsAccession # preference Reference alkene(s) NonA YP_001734428.1 C16:0,C18:0 Our C17:1, C19:1 results (WT) SafB AAU28294.1 C14:0, C16:0 Koketsuet C15:1, C17:1 al. 2010 MycA YP_003866245.1 C16:0 Hansen et C17:1 al.2007 DptE AAX31555.1 C12:0 Wittmann C13:1 et al. 2008

Example 4 Construction of Escherichia coli Comprising Recombinant NonA

The Synechococcus sp. PCC 7002 nonA (Genbank NC_(—)010475, locus A1173)was purchased from DNA 2.0. The sequence of nonA was codon optimized andoptimized for mRNA secondary structure. Unwanted restriction sites wereremoved from nonA and unique restriction sites flanking domains and N-and C-terminal Strep-tag II and His tags were added to the nonAsequence. The resulting gene and encoded protein sequence for thisoptimized gene (nonA_optV6) is given in SEQ ID NO: 23 and 24,respectively. The broad spectrum phosphopantetheinyl transferase sfp(Quadri et al. 1998, Genbank protein P39135.2) was purchased from DNA2.0 following codon optimization, checking for mRNA secondary structureeffects and removal of unwanted restriction sites (SEQ ID NO: 25). TheSynechococcus sp. PCC 7002 gene A2265 (SEQ ID NO: 37) (Genbank NC010475, locus A2265) was amplified from Synechococcus sp. PCC 7002genomic DNA using the Phusion high-fidelity PCR kit (New EnglandBiolabs) following the manufacturer's instructions and the PCR primersA2265 FP Sad (ggGAGCTCaaggaattatagttatgcgcaaaccctggttaga) (SEQ ID NO:32) and A2265 RP SbfI (ggCCTGCAGGttatagggactggatcgccagttttttctgct) (SEQID NO: 33). NonA_optV6 was cloned into the NdeI-MfeI and sfp was clonedinto the NcoI-EcoRI restriction sites of pCDFDuet-1 (Novagen) to yieldpJB1412. A2265 was cloned into the SacI-SbfI restriction sites ofpJB1412 to yield pJB1522. The NonA interior acyl-binding pocket (IABP)variants were generated by cloning in the respectiveexpression-optimized sequences from DptE, SafB and MycA (prepared by DNA2.0) into the AccI-HindIII restriction sites present in nonA_optV6 toyield nonA_dptE, nonA_safB and nonA_mycA, respectively. The gene andencoded protein sequence for these chimeric alkene synthases are givenin SEQ ID NOs: 26 through 31. The IABPs from nonA_dptE, nonA_safB andnonA_mycA were cloned into the NdeI-StuI restriction sites of nonA_optV6in pJB1522 to yield pJB1629, pJB1630 and pJB1639, respectively. Theplasmids containing the four nonA variants (pJB1522, pJB1629, pJB1630and pJB1639) and pCDFDuet-1 were transformed into chemically competentE. coli BL21 DE(3) (Invitrogen) following the manufacturer's directionsto generate strains JCC2157, JCC2358, JCC2375, and JCC2372 (Table 2).

TABLE 2 Engineered E. coli BL21 DE(3) strains investigated for theproduction of 1-alkenes. Strain Plasmid Genes JCC308 pCDFDuet-1 —JCC2157 pJB1522 sfp, nonA_optV6, A2265 JCC2358 pJB1629 sfp, nonA_dptE,A2265 JCC2375 pJB1630 sfp, nonA_safB, A2265 JCC2372 pJB1639 sfp,nonA_mycA, A2265

Example 5 Olefin Chain-Lengths Produced Via Expression of NonA-optV6 inEscherichia coli Culture Conditions and Sampling:

Single colonies of JCC308 and JCC2157 from LB plates containing 1%glucose and 50 mg/L spectinomycin were grown for 6 h at 37° C. in 4 mlof LB medium containing the same glucose and antibiotic concentration.These starter cultures were used to inoculate 15 ml cultures at astarting OD₆₀₀ of 0.05 in a 2% casamino acid M9-derived medium that wasamended to contain three times the M9 concentration of phosphate (33.9g/L Na₂HPO₄ and 9 g/L KH₂PO₄) and was supplemented with 3 mg/LFeSO₄.7H₂O, 0.01 mM IPTG and 50 mg/L spectinomycin. The cultures wereincubated for 68 h at 30° C./225 rpm in a New Brunswick shakingincubator. At this point, 50 μl of the cultures were removed todetermine the OD₆₀₀ and the remaining volume of the cultures (13 ml) waspelleted by centrifugation. The supernatant was discarded, the cellsresuspended in 1 ml of milli-Q water, transferred to a microcentrifugetube and pelleted by centrifugation. After removing any residual aqueousmedium, the cell pellets were vortexed for 20 seconds in 1 ml of acetone(Acros Organics 326570010) containing 25 mg/L butylated hydroxytoluene(antioxidant) and 25 mg/L eicosane (internal standard). The debris waspelleted by centrifugation and the acetone supernatants were analyzedfor the presence of 1-alkenes.

Identification and Quantification of 1-Alkenes

An Agilent 7890A GC/5975C EI-MS equipped with a 7683B autosampler wasused to identify the 1-alkenes. One μL of each sample was injected intothe GC inlet using pulsed splitless injection (pressure: 20 psi, pulsetime: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlettemperature of 290° C. The column was a HP-5MS-UI (Agilent, 20 m×0.18mm×0.18 μm) and the carrier gas was helium at a flow of 0.72 mL/min. TheGC oven temperature program was 80° C., hold 0.3 minute; 17.6°/minincrease to 290° C.; hold six minutes. The GC/MS interface was 290° C.,the MS mass range monitored was 25 to 400 amu and the temperatures ofthe source and quadrupole were 230° and 150° C., respectively.1-nonadecene (rt 8.4 min), 1-octadecene (rt 7.8) and 1-heptadecene (rt7.2 min) were identified based on comparison of their mass spectra (NISTMS database; 2008) and retention times with authentic standards. Shorterchain-length 1-alkenes were not detected in this experiment. The C19:21,x-nonadecadiene (rt 8.3) was identified based on interpretation of themass spectrum and a chemically consistent retention time. In someembodiments, 1,12-(cis)-nonadecadiene as cis-vaccenic acid is theprecursor for NonA to generate the nonadecadiene.

An Agilent 7890A GC/FID equipped with a 7683 series autosampler was usedto quantify the 1-alkenes. One μL of each sample was injected into theGC inlet (split 8:1, pressure: 20 psi, pulse time: 0.3 min, purge time:0.2 min, purge flow: 15 mL/min) which had an inlet temperature of 290°C. The column was a HP-5MS (Agilent, 20 m×0.18 mm×0.18 μm) and thecarrier gas was helium at a flow of 1.0 mL/min. The GC oven temperatureprogram was 80° C., hold 0.3 minute; 17.6°/min increase to 290° C.; hold6 minutes. Calibration curves were constructed for the detected1-alkenes using commercially available standards (Sigma-Aldrich), andthe concentrations of the 1-alkenes present in the extracts weredetermined based on the linear regressions of the peak areas andconcentrations. The concentration of 1-nonadecadiene in the samples wasdetermined using the calibration curve for 1-nonadecene. Theconcentrations of the compounds were normalized to the internal standard(eicosane) and reported as mg/L of culture.

The total ion count (TIC) chromatograms for JCC2157 and JCC308 are shownin FIG. 7. Four 1-alkenes are present in JCC2157 that are not found inJCC308. The mass spectra for the 1-alkenes and comparison with authenticstandards where possible are shown in FIG. 8. The quantification datafrom the experiment are summarized in Table 3.

TABLE 3 The optical densities of the cultures and the total mg/L of1-alkenes produced by the BL21 DE(3) strains. The % DCW was estimatedbased on the OD measurement using an average of 400 mg L⁻¹ OD₆₀₀ ⁻¹1-alkenes 1-alkenes (% of Strain OD₆₀₀ (mg/L) DCW) JCC308 2.7 — —JCC2157 3.2 0.28 0.022

Example 6 Production of Shorter Chain-Length 1-Alkenes with EngineeredAlkene Synthases Culture Conditions and Sampling:

Single colonies of JCC2157, JCC2358, JCC2375 and JCC2372 from LB platescontaining 50 mg/L spectinomycin were incubated for 18 h at 37° C. in 4ml of LB medium containing 50 mg/L spectinomycin. These starter cultureswere used to inoculate 15 ml cultures at a starting OD₆₀₀ of 0.05 in a2% glucose M9-derived medium that was amended to contain three times theM9 concentration of phosphate (33.9 g/L Na₂HPO₄ and 9 g/L KH₂PO₄) andwas supplemented with 3 mg/L FeSO₄.7H₂O, 0.01 mM IPTG and 50 mg/Lspectinomycin. The cultures were incubated for 54 h at 30° C./225 rpm ina New Brunswick shaking incubator. At this point, 50 μl of the cultureswere removed to determine the OD₆₀₀ and the remaining volume of thecultures (14 ml) was pelleted by centrifugation. The supernatant wasdiscarded, the cells resuspended in 1 ml of milli-Q water, transferredto a microcentrifuge tube and pelleted by centrifugation. After removingany residual aqueous medium, the cell pellets were vortexed for 20seconds in 1 ml of acetone (Acros Organics 326570010) containing 25 mg/Lbutylated hydroxytoluene (antioxidant) and 25 mg/L eicosane (internalstandard). The debris was pelleted by centrifugation and the acetonesupernatants were analyzed for the presence of 1-alkenes. The cellpellet extractions and GC analysis was performed as described in Example5.

Analysis of the GC chromatograms and quantification of peaks with thesame retention times as authentic standards revealed the presence ofshorter chain-length alkenes produced by some of the engineered alkenesynthases (Table 4). JCC2375 (nonA_safB) was particularly noteworthy asthe 1-alkenes produced were primarily 1-tridecene and 1-pentadecene asopposed to the longer chain length 1-alkenes detected from JCC2157bearing nonA_optV6 (FIG. 9). The mass spectra for the 1-tridecene peakin comparison with the authentic standard is shown in FIG. 10. Thisolefin (1-tridecene) is 4-6 methylene units shorter than the 1-alkenesproduced by the wild-type enzyme. This demonstrates that the chainlength specificity of these enzymes can be changed via tailoring oftheir acyl-binding pockets.

TABLE 4 The optical densities of the cultures and the mg/L of the1-alkenes produced by the BL21 DE(3) strains. Distribution of 1-alkenesin cells as mg/L of BL21 Total 1-alkenes culture strain IABP OD₆₀₀ (mg/Lof culture) C19:1 C19:2 C17:1 C15:1 C13:1 JCC2358 dptE 6.2 0.03 0.007 —0.027 — 0.008 JCC2375 safB 6.2 0.05 0.006 — 0.004 0.036 0.015 JCC2372mycA 5.9 0.04 0.009 0.009 0.026 — 0.009 JCC2157 nonA 6.1 0.39 0.1530.088 0.149 0.004 —

Complete cites to various articles referred to herein are providedbelow:

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All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties and for allpurposes.

INFORMAL SEQUENCE LISTING SEQ ID NO: 1 Nucleotide nonA>SYNPCC7002_A1173 polyketide synthase (PKS) [Synechococcus sp. PCC 7002Interior Acyl Binding Pocket underlinedATGGTTGGTCAATTTGCAAATTTCGTCGATCTGCTCCAGTACAGAGCTAAACTTCAGGCGCGGAAAACCGTGTTTAGTTTTCTGGCTGATGGCGAAGCGGAATCTGCGGCCCTGACCTACGGAGAATTAGACCAAAAAGCCCAGGCGATCGCCGCTTTTTTGCAAGCTAACCAGGCTCAAGGGCAACGGGCATTATTACTTTATCCACCGGGTTTAGAGTTTATCGGTGCCTTTTTGGGATGTTTGTATGCTGGTGTTGTTGCGGTGCCAGCTTACCCACCACGGCCGAATAAATCCTTTGACCGCCTCCATAGCATTATCCAAGATGCCCAGGCAAAATTTGCCCTCACCACAACAGAACTTAAAGATAAAATTGCCGATCGCCTCGAAGCTTTAGAAGGTACGGATTTTCATTGTTTGGCTACAGATCAAGTTGAATTAATTTCAGGAAAAAATTGGCAAAAACCGAACATTTCCGGCACAGATCTCGCTTTTTTGCAATACACCAGTGGCTCCACGGGCGATCCTAAAGGAGTGATGGTTTCCCACCACAATTTGATCCACAACTCCGGCTTGATTAACCAAGGATTCCAGGATACAGAGGCGAGTATGGGCGTTTCCTGGTTGCCGCCCTACCATGATATGGGCTTGATCGGTGGGATTTTACAGCCCATCTATGTGGGAGCAACGCAAATTTTAATGCCTCCCGTGGCCTTTTTGCAGCGACCTTTTCGGTGGCTAAAGGCGATCAACGATTATCGGGTTTCCACCAGCGGTGCGCCGAATTTTGCCTATGATCTCTGTGCCAGCCAAATTACCCCGGAACAAATCAGAGAACTCGATTTGAGCTGTTGGCGACTGGCTTTTTCCGGGGCCGAACCGATCCGCGCTGTGACCCTCGAAAATTTTGCGAAAACCTTCGCTACAGCAGGCTTTCAAAAATCAGCATTTTATCCCTGTTATGGTATGGCTGAAACCACCCTGATCGTTTCCGGTGGTAATGGTCGTGCCCAGCTTCCCCAGGAAATTATCGTCAGCAAACAGGGCATCGAAGCAAACCAAGTTCGCCCTGCCCAAGGGACAGAAACAACGGTGACCTTGGTCGGCAGTGGTGAAGTGATTGGCGACCAAATTGTCAAAATTGTTGACCCCCAGGCTTTAACAGAATGTACCGTCGGTGAAATTGGCGAAGTATGGGTTAAGGGCGAAAGTGTTGCCCAGGGCTATTGGCAAAAGCCAGACCTCACCCAGCAACAATTCCAGGGAAACGTCGGTGCAGAAACGGGCTTTTTACGCACGGGCGATCTGGGTTTTTTGCAAGGTGGCGAACTGTATATTACGGGTCGTTTAAAGGATCTCCTGATTATCCGGGGGCGCAACCACTATCCCCAGGACATTGAATTAACCGTCGAAGTGGCCCATCCCGCTTTACGACAGGGGGCCGGAGCCGCTGTATCAGTAGACGTTAACGGGGAAGAACAGTTAGTCATTGTCCAGGAAGTTGAGCGTAAATATGCCCGCAAATTAAATGTCGCGGCAGTAGCCCAAGCTATTCGTGGGGCGATCGCCGCCGAACATCAACTGCAACCCCAGGCCATTTGTTTTATTAAACCCGGTAGCATTCCCAAAACATCCAGCGGGAAGATTCGTCGCCATGCCTGCAAAGCTGGTTTTCTAGACGGAAGCTTGGCTGTGGTTGGGGAGTGGCAACCCAGCCACCAAAAAGAAGGAAAAGGAATTGGGACACAAGCCGTTACCCCTTCTACGACAACATCAACGAATTTTCCCCTGCCTGACCAGCACCAACAGCAAATTGAAGCCTGGCTTAAGGATAATATTGCCCATCGCCTCGGCATTACGCCCCAACAATTAGACGAAACGGAACCCTTTGCAAGTTATGGGCTGGATTCAGTGCAAGCAGTACAGGTCACAGCCGACTTAGAGGATTGGCTAGGTCGAAAATTAGACCCCACTCTGGCCTACGATTATCCGACCATTCGCACCCTGGCTCAGTTTTTGGTCCAGGGTAATCAAGCGCTAGAGAAAATACCACAGGTGCCGAAAATTCAGGGCAAAGAAATTGCCGTGGTGGGTCTCAGTTGTCGTTTTCCCCAAGCTGACAACCCCGAAGCTTTTTGGGAATTATTACGTAATGGTAAAGATGGAGTTCGCCCCCTTAAAACTCGCTGGGCCACGGGAGAATGGGGTGGTTTTTTAGAAGATATTGACCAGTTTGAGCCGCAATTTTTTGGCATTTCCCCCCGGGAAGCGGAACAAATGGATCCCCAGCAACGCTTACTGTTAGAAGTAACCTGGGAAGCCTTGGAACGGGCAAATATTCCGGCAGAAAGTTTACGCCATTCCCAAACGGGGGTTTTTGTCGGCATTAGTAATAGTGATTATGCCCAGTTGCAGGTGCGGGAAAACAATCCGATCAATCCCTACATGGGGACGGGCAACGCCCACAGTATTGCTGCGAATCGTCTGTCTTATTTCCTCGATCTCCGGGGCGTTTCTCTGAGCATCGATACGGCCTGTTCCTCTTCTCTGGTGGCGGTACATCTGGCCTGTCAAAGTTTAATCAACGGCGAATCGGAGTTGGCGATCGCCGCCGGGGTGAATTTGATTTTGACCCCCGATGTGACCCAGACTTTTACCCAGGCGGGCATGATGAGTAAGACGGGCCGTTGCCAGACCTTTGATGCCGAGGCTGATGGCTATGTGCGGGGCGAAGGTTGTGGGGTCGTTCTCCTCAAACCCCTGGCCCAGGCAGAACGGGACGGGGATAATATTCTCGCGGTGATCCACGGTTCGGCGGTGAATCAAGATGGACGCAGTAACGGTTTGACGGCTCCCAACGGGCGATCGCAACAGGCCGTTATTCGCCAAGCCCTGGCCCAAGCCGGCATTACCGCCGCCGATTTAGCTTACCTAGAGGCCCACGGCACCGGCACGCCCCTGGGTGATCCCATTGAAATTAATTCCCTGAAGGCGGTTTTACAAACGGCGCAGCGGGAACAGCCCTGTGTGGTGGGTTCTGTGAAAACAAACATTGGTCACCTCGAGGCAGCGGCGGGCATCGCGGGCTTAATCAAGGTGATTTTGTCCCTAGAGCATGGAATGATTCCCCAACATTTGCATTTTAAGCAGCTCAATCCCCGCATTGATCTAGACGGTTTAGTGACCATTGCGAGCAAAGATCAGCCTTGGTCAGGCGGGTCACAAAAACGGTTTGCTGGGGTAAGTTCCTTTGGGTTTGGTGGCACCAATGCCCACGTGATTGTCGGGGACTATGCTCAACAAAAATCTCCCCTTGCTCCTCCGGCTACCCAAGACCGCCCTTGGCATTTGCTGACCCTTTCTGCTAAAAATGCCCAGGCCTTAAATGCCCTGCAAAAAAGCTATGGAGACTATCTGGCCCAACATCCCAGCGTTGACCCACGCGATCTCTGTTTGTCTGCCAATACCGGGCGATCGCCCCTCAAAGAACGTCGTTTTTTTGTCTTTAAACAAGTCGCCGATTTACAACAAACTCTCAATCAAGATTTTCTGGCCCAACCACGCCTCAGTTCCCCCGCAAAAATTGCCTTTTTGTTTACGGGGCAAGGTTCCCAATACTACGGCATGGGGCAACAACTGTACCAAACCAGCCCAGTATTTCGGCAAGTGCTGGATGAGTGCGATCGCCTCTGGCAGACCTATTCCCCCGAAGCCCCTGCCCTCACCGACCTGCTGTACGGTAACCATAACCCTGACCTCGTCCACGAAACTGTCTATACCCAGCCCCTCCTCTTTGCTGTTGAATATGCGATCGCCCAACTATGGTTAAGCTGGGGCGTGACGCCAGACTTTTGCATGGGCCATAGCGTCGGCGAATATGTCGCGGCTTGTCTGGCGGGGGTATTTTCCCTGGCAGACGGCATGAAATTAATTACGGCCCGGGGCAAACTGATGCACGCCCTACCCAGCAATGGCAGTATGGCGGCGGTCTTTGCCGATAAAACGGTCATCAAACCCTACCTATCGGAGCATTTGACCGTCGGAGCCGAAAACGGTTCCCATTTGGTGCTATCAGGAAAGACCCCCTGCCTCGAAGCCAGTATTCACAAACTCCAAAGCCAAGGGATCAAAACCAAACCCCTCAAGGTTTCCCATGCTTTCCACTCCCCTTTGATGGCTCCCATGCTGGCAGAGTTTCGGGAAATTGCTGAACAAATTACTTTCCACCCGCCGCGTATCCCGCTCATTTCCAATGTCACGGGCGGCCAGATTGAAGCGGAAATTGCCCAGGCCGACTATTGGGTTAAGCACGTTTCGCAACCCGTCAAATTTGTCCAGAGCATCCAAACCCTGGCCCAAGCGGGTGTCAATGTTTATCTCGAAATCGGCGTAAAACCAGTGCTCCTGAGTATGGGACGCCATTGCTTAGCTGAACAAGAAGCGGTTTGGTTGCCCAGTTTACGTCCCCATAGTGAGCCTTGGCCGGAAATTTTGACCAGTCTCGGCAAACTGTATGAGCAAGGGCTAAACATTGACTGGCAGACCGTGGAAGCTGGCGATCGCCGCCGGAAACTGATTCTGCCCACCTATCCCTTCCAACGGCAACGATATTGGTTTAATCAAGGCTCTTGGCAAACTGTTGAGACCGAATCTGTGAACCCAGGCCCTGACGATCTCAATGATTGGTTGTATCAGGTGGCGTGGACGCCCCTGGACACTTTGCCCCCGGCCCCTGAACCGTCGGCTAAGCTGTGGTTAATCTTGGGCGATCGCCATGATCACCAGCCCATTGAAGCCCAATTTAAAAACGCCCAGCGGGTGTATCTCGGCCAAAGCAATCATTTTCCGACGAATGCCCCCTGGGAAGTATCTGCCGATGCGTTGGATAATTTATTTACTCACGTCGGCTCCCAAAATTTAGCAGGCATCCTTTACCTGTGTCCCCCAGGGGAAGACCCAGAAGACCTAGATGAAATTCAAAAGCAAACCAGTGGCTTCGCCCTCCAACTGATCCAAACCCTGTATCAACAAAAGATCGCGGTTCCCTGCTGGTTTGTGACCCACCAGAGCCAACGGGTGCTTGAAACCGATGCTGTCACCGGATTTGCCCAAGGGGGATTATGGGGACTCGCCCAGGCGATCGCCCTCGAACATCCAGAGTTGTGGGGGGGAATTATTGATGTCGATGACAGCCTGCCAAATTTTGCCCAGATTTGCCAACAAAGACAGGTGCAGCAGTTGGCCGTGCGGCACCAAAAACTCTACGGGGCACAGCTCAAAAAGCAACCGTCACTGCCCCAGAAAAATCTCCAGATTCAACCCCAACAGACCTATCTAGTGACAGGGGGACTGGGGGCCATTGGCCGTAAAATTGCCCAATGGCTAGCCGCAGCAGGAGCAGAAAAAGTAATTCTCGTCAGCCGGCGCGCTCCGGCAGCGGATCAGCAGACGTTACCGACCAATGCGGTGGTTTATCCTTGCGATTTAGCCGACGCAGCCCAGGTGGCAAAGCTGTTTCAAACCTATCCCCACATCAAAGGAATTTTCCATGCGGCGGGTACCTTAGCTGATGGTTTGCTGCAACAACAAACTTGGCAAAAGTTCCAGACCGTCGCCGCCGCCAAAATGAAAGGGACATGGCATCTGCACCGCCATAGTCAAAAGCTCGATCTGGATTTTTTTGTGTTGTTTTCCTCTGTGGCAGGGGTGCTCGGTTCACCGGGACAGGGGAATTATGCCGCCGCAAACCGGGGCATGGCGGCGATCGCCCAATATCGACAAGCCCAAGGTTTACCCGCCCTGGCGATCCATTGGGGGCCTTGGGCCGAAGGGGGAATGGCCAACTCCCTCAGCAACCAAAATTTAGCGTGGCTGCCGCCCCCCCAGGGACTAACAATCCTCGAAAAAGTCTTGGGCGCCCAGGGGGAAATGGGGGTCTTTAAACCGGACTGGCAAAACCTGGCCAAACAGTTCCCCGAATTTGCCAAAACCCATTACTTTGCAGCCGTTATTCCCTCTGCTGAGGCTGTGCCCCCAACGGCTTCAATTTTTGACAAATTAATCAACCTAGAAGCTTCTCAGCGGGCTGACTATCTACTGGATTATCTGCGGCGGTCTGTGGCGCAAATCCTCAAGTTAGAAATTGAGCAAATTCAAAGCCACGATAGCCTGTTGGATCTGGGCATGGATTCGTTGATGATCATGGAGGCGATCGCCAGCCTCAAGCAGGATTTACAACTGATGTTGTACCCCAGGGAAATCTACGAACGGCCCAGACTTGATGTGTTGACGGCCTATCTAGCGGCGGAATTCACCAAGGCCCATGATTCTGAAGCAGCAACGGCGGCAGCAGCGATTCCCTCCCAAAGCCTTTCGGTCAAAACAAAAAAACAGTGGCAAAAACCTGACCACAAAAACCCGAATCCCATTGCCTTTATCCTCTCTAGCCCCCGGTCGGGTTCGACGTTGCTGCGGGTGATGTTAGCCGGACATCCGGGGTTATATTCGCCGCCAGAGCTGCATTTGCTCCCCTTTGAGACTATGGGCGATCGCCACCAGGAATTGGGTCTATCCCACCTCGGCGAAGGGTTACAACGGGCCTTAATGGATCTAGAAAACCTCACCCCAGAGGCAAGCCAGGCGAAGGTCAACCAATGGGTCAAAGCGAATACACCCATTGCAGACATCTATGCCTATCTCCAACGGCAGGCGGAACAACGTTTACTCATCGACAAATCTCCCAGCTACGGCAGCGATCGCCATATTCTAGACCACAGCGAAATCCTCTTTGACCAGGCCAAATATATCCATCTGGTACGCCATCCCTACGCGGTGATTGAATCCTTTACCCGACTGCGGATGGATAAACTGCTGGGGGCCGAGCAGCAGAACCCCTACGCCCTCGCGGAGTCCATTTGGCGCACCAGCAACCGCAATATTTTAGACCTGGGTCGCACGGTTGGTGCGGATCGATATCTCCAGGTGATTTACGAAGATCTCGTCCGTGACCCCCGCAAAGTTTTGACAAATATTTGTGATTTCCTGGGGGTGGACTTTGACGAAGCGCTCCTCAATCCCTACAGCGGCGATCGCCTTACCGATGGCCTCCACCAACAGTCCATGGGCGTCGGGGATCCCAATTTCCTCCAGCACAAAACCATTGATCCGGCCCTCGCCGACAAATGGCGCTCAATTACCCTGCCCGCTGCTCTCCAGCTGGATACGATCCAGTTGGCCGAAACGTTTGCTTACGATCTCCCCCAGGAACCCCAGCTAACACCCCAGACCCAATCCTTGCCCTCGATGGTGGAGCGGTTCGTGACAGTGCGCGGTTTAGAAACCTGTCTCTGTGAGTGGGGCGATCGCCACCAACCATTGGTGCTACTTCTCCACGGCATCCTCGAACAGGGGGCCTCCTGGCAACTCATCGCGCCCCAGTTGGCGGCCCAGGGCTATTGGGTTGTGGCCCCAGACCTGCGTGGTCACGGCAAATCCGCCCATGCCCAGTCCTACAGCATGCTTGATTTTTTGGCTGACGTAGATGCCCTTGCCAAACAATTAGGCGATCGCCCCTTTACCTTGGTGGGCCACTCCATGGGTTCCATCATCGGTGCCATGTATGCAGGAATTCGCCAAACCCAGGTAGAAAAGTTGATCCTCGTTGAAACCATTGTCCCCAACGACATCGACGACGCTGAAACCGGTAATCACCTGACGACCCATCTCGATTACCTCGCCGCGCCCCCCCAACACCCGATCTTCCCCAGCCTAGAAGTGGCCGCCCGTCGCCTCCGCCAAGCCACGCCCCAACTACCCAAAGACCTCTCGGCGTTCCTCACCCAGCGCAGCACCAAATCCGTCGAAAAAGGGGTGCAGTGGCGTTGGGATGCTTTCCTCCGTACCCGGGCGGGCATTGAATTCAATGGCATTAGCAGACGACGTTACCTGGCCCTGCTCAAAGATATCCAAGCGCCGATCACCCTCATCTATGGCGATCAGAGTGAATTTAACCGCCCTGCTGATCTCCAGGCGATCCAAGCGGCTCTCCCCCAGGCCCAACGTTTAACGGTTGCTGGCGGCCATAACCTCCATTTTGAGAATCCCCAGGCGATCGCCCAAATTGTTTATCAACAACTCCAGACCCCTGTACCCAAAACACAATAA SEQ ID NO: 2 Amino Acid NonA>gi|170077790|ref|YP_001734428.1|polyketide synthase [Synechococcus sp. PCC 7002]Interior Acyl Binding Pocket underlinedMVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLINQGFQDTEASMGVSWLPPYHDMGLIGGILQPIYVGATQILMPPVAFLQRPFRWLKAINDYRVSTSGAPNFAYDLCASQITPEQIRELDLSCWRLAFSGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQSEQ ID NO: 3: Nucleotide IABP (Interior Acyl Binding Pocket) of nonAATTAACCAAGGATTCCAGGATACAGAGGCGAGTATGGGCGTTTCCTGGTTGCCGCCCTACCATGATATGGGCTTGATCGGTGGGATTTTACAGCCCATCTATGTGGGAGCAACGCAAATTTTAATGCCTCCCGTGGCCTTTTTGCAGCGACCTTTTCGGTGGCTAAAGGCGATCAACGATTATCGGGTTTCCACCAGCGGTGCGCCGAATTTTGCCTATGATCTCTGTGCCAGCCAAATTACCCCGGAACAAATCAGAGAACTCGATTTGAGCTGTTGGCGACTGGCTTTTTCCSEQ ID NO: 4: Amino Acid IABP (Interior Acyl Binding Pocket) of NonAINQGFQDTEASMGVSWLPPYHDMGLIGGILQPIYVGATQILMPPVAFLQRPFRWLKAINDYRVSTSGAPNFAYDLCSQITPEQIRELDLSCWRLAFS SEQ ID NO: 5 NucleotideIpg2229 saframycin Mx1 synthetase B (safB)>gi|52840256: 25|9995-2521740 Legionella pneumophila subsp. pneumophila str.Philadelphia 1 chromosome, complete genomeInterior Acyl Binding Pocket underlinedGTGAAAAAAGAATATTTGCAGTGCCAGTCTCTGGTTGACGTCGTCAGGTTACGTGCTTTACACAGCCCTAACAAGAAAAGCTGTACTTTTCTGAACAAAGAGTTGGAAGAGACGATGACTTATGAGCAACTGGATCAACACGCCAAAGCAATTGCGGCAACTTTGCAAGCAGAAGGAGCAAAACCTGGGGATAGGGTCTTGTTATTGTTTGCACCTGGATTACCCCTTATCCAGGCATTTTTGGGCTGCCTTTATGCAGGCTGCATTGCTGTACCCATTTACCCACCAGCCCAAGAAAAATTATTGGACAAGGCACAACGCATAGTTACCAACTCAAAACCGGTCATAGTACTGATGATTGCGGATCACATCAAAAAATTCACCGCAGACGAATTAAATACAAATCCCAAATTCCTGAAAATTCCTGCTATTGCGCTTGAGAGCATTGAGTTAAACAGAAGTAGTAGTTGGCAACCAACCTCCATTAAGAGCAATGACATTGCGTTTCTGCAATACACTTCCGGCTCAACCATGCACCCTAAAGGAGTGATGGTAAGCCACCATAATTTACTGGATAATCTGAATAAS ATTTTTACCTCTTTTCATATGAATGATGAAACCATTATTTTCAGCTGGCTGCCCCCACATCATGATATGGGTTTGATTGGCTGCATTCTGACCCCCATCTATGGTGGAATTCAGGCAATCATGATGTCCCCTTTCTCATTTTTACAAAACCCGCTTTCCTGGTTAAAACATATTACCAAATACAAAGCAACTATCAGTGGAAGCCCTAACTTCGCTTACGATTATTGTGTCAAACGAATCAGGGAAGAAAAAAAAGAAGGGCTGGATTTAAGTTCATGGGTGACTGCTTTCAAC GGTGCTGAGCCAGTACGAGAAGAAACCATGGAACATTTTTATCAGGCATTTAAAGAGTTTGGATTTCGTAAAGAAGCCTTCTATCCATGCTATGGCCTGGCTGAGGCCACTTTGTTAGTGACGGGAGGAACACCAGGAAGTTCATACAAAACATTAACTCTGGCCAAAGAACAATTTCAAGATCATCGCGTGCATTTTGCAGACGATAACAGTCCAGGCAGTTACAAGTTAGTCAGCAGTGGTAATCCCATTCAAGAAGTTAAAATTATAGATCCTGATACCTTGATCCCATGTGATTTTGACCAGGTTGGTGAAATTTGGGTACAAAGTAACAGTGTCGCCAAAGGATATTGGAATCAACCCGAAGAAACAAGGCATGCGTTCGCAGGAAAAATTAAAGACGATGAGCGTAGCGCAATCTATTTAAGAACCGGGGACTTGGGCTTTCTCCATGAAAATGAGTTATACGTTACTGGACGCATTAAAGACTTAATTATTATTTATGGTAAAAATCATTATCCTCAGGACATAGAGTTCAGCCTGATGCATTCTCCGCTCCATCACGTATTGGGCAAATGCGCTGCTTTTGTGATTCAGGAGGAGCATGAATATAAACTGACTGTGATGTGTGAAGTAAAAAATCGATTCATGGATGACGTAGCTCAAGACAATTTATTCAATGAGATTTTTGAGCTTGTTTACGAAAACCACCAATTGGAGGTACATACTATAGTCCTGATTCCTCTTAAAGCAATGCCACATACTACCAGCGGAAAAATTCGCAGGAATTTTTGTCGAAAACATCTTTTGGATAAAACTCTGCCAATAGTGGCTACCTGGCAACTCAATAAAATTGAGGAATAASEQ ID NO: 6 Amino AcidSaframycin Mx1 synthetase B (Legionella pneumophila) SafB AAU28294.1Interior Acyl Binding Pocket domain underlined        10         20         30         40         50         60MKKEYLQCQS LVDVVRLRAL HSPNKKSCTF LNKELEETMT YEQLDQHAKA IAATLQAEGA        70         80         90        100        110        120KPGDRVLLLF APGLPLIQAF LGCLYAGCIA VPIYPPAQEK LLDKAQRIVT NSKPVIVLMI       130        140        150        160        170        180ADHIKKFTAD ELNTNPKFLK IPAIALESIE LNRSSSWQPT SIKSNDIAFL QYTSGSTMHP       190        200        210        220        230        240KGVMVSHHNL LDNLNKIFTS FHMNDETIIF SWLPPHHDMG LIGCILTPIY GGIQAIMMSP       250        260        270        280        290        300FSFLQNPLSW LKHITKYKAT ISGSPNFAYD YCVKRIREEK KEGLDLSSWV TAFNGAEPVR       310        320        330        340        350        360EETMEHFYQA FKEFGFRKEA FYPCYGLAEA TLLVTGGTPG SSYKTLTLAK EQFQDHRVHF       370        380        390        400        410        420ADDNSPGSYK LVSSGNPIQE VKIIDPDTLI PCDFDQVGEI WVQSNSVAKG YWNQPEETRH       430        440        450        460        470        480AFAGKIKDDE RSAIYLRTGD LGFLHENELY VTGRIKDLII IYGKNHYPQD IEFSLMHSPL       490        500        510        520        530        540HHVLGKCAAF VIQEEHEYKL TVMCEVKNRF MDDVAQDNLF NEIFELVYEN HQLEVHTIVL       550        560        570        580IPLKAMPHTT SGKIRRNFCR KHLLDKTLPI VATWQLNKIE E SEQ ID NO: 7 NucleotideInterior Acyl Binding Pocket of safBATTTTTACCTCTTTTCATATGAATGATGAAACCATTATTTTCAGCTGGCTGCCCCCACATCATGATATGGGTTTGATTGGCTGCATTCTGACCCCCATCTATGGTGGAATTCAGGCAATCATGATGTCCCCTTTCTCATTTTTACAAAACCCGCTTTCCTGGTTAAAACATATTACCAAATACAAAGCAACTATCAGTGGAAGCCCTAACTTCGCTTACGATTATTGTGTCAAACGAATCAGGGAAGAAAAAAAAGAAGGGCTGGATTTAAGTTCATGGGTGACTGCTTTCAACSEQ ID NO: 8 Amino Acid Interior Acyl Binding Pocket of SafBIFTSFHMNDETIIFSWLPPHHDMGLIGCILTPIYGGIQAIMMSPFSFLQNPLSWLKHITKYKATISGSPNFAYDYCVKRIREEKKEGLDLSSWVTAFN SEQ ID NO: 9 NucleotideMycosubtilin synthase subunit mycA(YP_003866245.1)>gi|305672698: c1947930-1936015 Bacillus subtilis subsp. spizizenii str. W23chromosome, complete genomeInterior Acyl Binding Pocket domain underlinedATGTATACCAGTCAATTTCAAACCTTAGTCGATGTTATTCGGAATAGAAGCAATATATCAGATCGCGGGATCCGTTTTATCGAATCCGATAAAATCGAGACATTTGTCTCTTATCGCCAATTGTTTGACGAGGCGCAAGGTTTTCTTGGCTACTTACAACATATCGGCATTCAGCCAAAGCAAGAAATTGTGTTTCAAATTCAAGAAAACAAATCATTTGTCGTCGCTTTTTGGGCGTGTTTATTAGGAGGAATGATTCCGGTACCCGTCAGTATCGGAGAAGATAATGACCACAAGCTAAAGGTATGGCGCATTTGGAATATTTTAAACAATCCATTCTTGCTAGCCTCTGAAACAGTATTAGATAAAATGAAAAAATTTGCTGCTGATCACGATTTACAAGATTTCCATCATCAATTAATCGAGAAATCCGACATCATTCAGGATCGAATCTACGATCACCCGGCTTCGCAATATGAACCTGAAGCCGATGAATTGGCCTTTATTCAATTTTCTTCGGGATCAACAGGAGACCCGAAAGGAGTCATGCTAACCCATCATAACTTAATACATAATACATGTGCAATCCGGAATGCGCTGGCTATCGACTTAAAAGATACTCTTTTATCTTGGATGCCCTTAACCCATGACATGGGGCTCATAGCTTGCCACCTTGTTCCTGCCTTAGCCGGAATCAATCAAAATTTAATGCCGACAGAATTATTTATTCGAAGACCTATTCTCTGGATGAAAAAAGCTCATGAACATAAAGCCAGCATTCTATCCTCACCTAATTTTGGATACAATTACTTTCTTAAATTTCTGAAAGACAATAAAAGTTACGACTGGGATTTATCCCATATCAGGGTCATTGCAAACGGAGCAGAACCTATATTGCCAGAGCTATGTGATGAATTTTTGACTAGATGCGCAGCATTCAATATGAAACGATCTGCCATCTTAAATGTTTACGGTTTAGCTGAGGCTTCGGTTGGCGCAACATTCTCTAACATCGGAGAAAGATTTGTCCCTGTTTATTTGCATCGCGATCATCTAAATCTAGGTGAAAGAGCCGTTGAAGTAAGCAAAGAGGATCAAAATTGCGCTTCATTCGTCGAAGTAGGAAAGCCTATTGATTACTGCCAAATTCGAATCTGTAATGAAGCAAACGAAGGATTGGAAGACGGATTTATCGGTCATATCCAGATCAAGGGGGAGAATGTGACCCAAGGGTATTATAACAACCCCGAAAGTACGAACAGAGCGCTGACTCCCGATGGATGGGTGAAAACGGGAGATCTTGGCTTCATTAGAAAAGGGAATTTAGTCGTAACCGGAAGGGAAAAAGACATTATTTTTGTGAACGGAAAAAATGTGTATCCTCACGATATTGAACGAGTCGCCATTGAATTAGAGGACATTGATTTAGGAAGAGTTGCAGCCTGTGGTGTATATGATCAAGAGACACGAAGCAGAGAAATTGTACTTTTTGCTGTTTACAAAAAATCAGCGGAGCAGTTTGCACCACTTGTTAAAGACATTAAAAAGCATTTGTACCAGCGAGGCGGATGGAGCATCAAAGAAATCCTGCCGATCCGAAAGCTGCCAAAAACGACAAGCGGGAAAGTTAAACGCTATGAGCTGGCTGAGCAGTATGAGTCGGGGAAATTTGCGCTAGAGTCAACCAAAATCAAGGAATTTTTGGAGGGTCATTCGACGGAACCGGTACAGACTCCTATTCATGAAATCGAAACAGCATTGCTGTCTATCTTTTCAGAAGTGATGGATGGAAAAAAGATTCACCTAAATGATCATTATTTTGACATGGGTGCAACCTCATTACAGTTATCTCAAATTGCCGAACGCATTGAACAAAAGTTTGGTTGTGAGCTTACGGTTGCTGATCTCTTTACATATCCTTCAATCGCTGATTTAGCGGCATTCCTTGTCGAAAACCATTCCGAAATCAAGCAAACTGATACAGCGAAGCCAAGCCGCTCTTCGTCAAAAGACATCGCTATTATCGGGATGTCCCTCAATGTTCCAGGGGCATCGAATAAGAGTGATTTTTGGCACCTGCTCGAAAACGGTGAGCATGGCATTCGGGAATATCCTGCACCAAGAGTTAAAGATGCGATAGATTATTTACGATCCATTAAAAGCGAACGTAACGAAAAACAATTTGTGAAGGGCGGCTATTTAGATGAGATAGACCGTTTTGATTATTCGTTCTTTGGTTTAGCTCCCAAAACCGCAAAATTCATGGATCCCAATCAAAGGCTATTTTTGCAATCCGCATGGCATGCGATTGAAGATGCAGGCTATGCCGGCGACACCATTAGCGGGAGTCAGCTCGGGGTATATGTAGGGTACTCGAAGGTGGGATACGATTACGAACGTCTCCTTTCTGCGAATTATCCGGAGGAGCTTCATCATTATATTGTGGGCAATCTTCCCTCGGTGTTGGCGAGTCGAATTGCTTACTTTCTAAATTTGAAAGGACCAGCGGTTACCGTGGATACAGCTTGCTCTTCGTCACTTGTTGCTGTTCATATGGCATGTAAAGCTTTGCTTACAGGCGATTGTGAAATGGCTCTTGCCGGGGGTATTCGAACTTCGCTATTACCGATGCGTATCGGTCTCGATATGGAATCTTCTGACGGGCTCACGAAAACGTTCAGCAAGGATTCGGACGGAACAGGCTCTGGCGAAGGCGTGGCAGCAGTCCTGTTGAAACCTTTGCAGGCTGCGATTCGCGATGGAGATCATATTTATGGTGTGATCAAGGGAAGCGCGATAAACCAAGACGGGACAACCGTTGGAATCACCGCACCGAGCCCGGCAGCTCAGACCGAGGTGATTGAGATGGCCTGGAAAGACGCTGGCATTGCTCCTGAAACATTGTCTTTCATTGAAGCACACGGCACCGGAACCAAGCTCGGGGATCCTGTTGAATTTAACGGTCTTTGTAAAGCGTTTGAGAAGGTTACGGAAAAGAAACAGTTTTGTGCGATCGGCTCTGTTAAAGCAAACATCGGTCATTTGTTTGAAGCGGCAGGCATCGTTGGACTGATAAAATCTGCCCTTATGTTGAATCACAAAAAAATCCCGCCGCTGGCTCACTTTAATAAACCGAATCCATTAATTCCATTTCACTCTTCTCCTTTTTATGTGAACCAAGAAGTGATGGATTTCACACCTGAAGACCGACCGCTGCGGGGTGGTATCAGTTCATTCGGTTTTAGCGGAACGAATGCCCATGTAGTATTGGAAGAATATACTCCTGAAAGTGAGTATGCACCGGAGGACGGGAATGATCCGCATTTATTTGTGTTATCCGCCCATACTGAAGCTTCACTATATGAACTGACTCATCAGTATCGGCAATATATTTCAGATGACAGCCAATCATCATTGAGGTCAATTTGCTATACGGCCAGTACAGGAAGGGCTCATTTAGATTATTGTTTAGCCATGATTGTATCCAGCAACCAAGAATTAATAGATAAGCTGACCAGTTTGATTCAAGGCGAAAGAAATCTTCCCCAAGTACACTTTGGCTATAAAAACATCAAGGAAATGCAGCCTGCCGAAAAAGACAATCTGAGTAAACAAATCTCTGATCTCATGCAGCATCGGCCCTGCACAAAGGATGAACGAATCACATGGTTGAATCGTATTGCAGAATTATATGTGCAAAGAGCCGTGATTGACTGGCGAGCGGTTTATTCCAATGAAGTTGTACAAAAAACGCCATTGCCTTTGTATCCATTTGAACGGAATCGCTGTTGGGTTGAGGCTGTCTATGAAAGCGCCAAGGAAAGAAAAGAGAAAGGGGAAGTAGCATTGGATATAAATCATACGAAGACACATATTGAGTCCTTTCTGAAGACTGTCATCAGCAATGCATCGGGAATCAGAGCGGATGAGATCGATTCGAATGCCCATTTTATCGGATTCGGATTGGATTCCATTATGCTGACACAGGTCAAAAAAGCGATCGCAGACGAATTTAATGTGGATATCCCGATGGAACGTTTTTTTGATACGATGAACAACATTGAAAGTGTTGTCGATTATTTGGCAGAAAATGTTCCATCAGCTGCATCCACTCCGCCTCAAGAAAGTGTTACGGCACAGGAAGAGCTTGTGATATCAGGAGCACAGCCCGAGTTGGAACATCAAGAGCATATGTTGGACAAAATTATTGCTTCTCAGAATCAATTAATCCAGCAAACTTTACAAGCTCAATTGGATAGCTTTAATTTGTTGAGAAACAACAGCCATTTTGTATCGAAAGAATCCGAGATTTCGCAAGATAAAACGAGCCTTTCTCCTAAATCTGTCACTGCAAAAAAGAATTCGGCTCAAGAAGCAAAACCTTATATTCCTTTTCAGCGTCAGACCTTGAATGAACAGGTCAACTATACTCCGCAGCAAAGACAATATTTAGAATCATTTATAGAGAAATACGTAGACAAAACGAAAGGTTCCAAGCAATATACGGACGAAACCCGATTTGCTCATGCCAATAACCGCAACTTGTCCAGCTTCCGGTCTTATTGGAAAGAAATGGTTTACCCGATCATCGCTGAACGCTCGGACGGTTCTAGAATGTGGGATATTGATGGAAATGAATATATCGATATCACCATGGGATTTGGGGTTAATCTTTTTGGGCATCACCCGTCCTTTATTACTCAAACCGTCGTTGATTCAACACATTCTGCATTGCCGCCTCTTGGTCCGATGTCAAATGTCGCCGGAGAAGTTGCAGATCGAATTCGTGCATGCACAGGAGTAGAAAGGGTCGCTTTTTATAATTCAGGCACGGAGGCAGTCATGGTTGCCCTGCGTTTGGCGAGGGCGGCAACAGGAAGAACGAAAGTGGTAGTGTTTGCGGGCTCTTATCACGGCACCTTTGACGGCGTATTAGGTGTTGCCAACACAAAAGGCGGGGCTGAGCCTGCGAATCCGCTGGCTCCGGGCATACCGCAAAGCTTTATGAATGATTTGATTATTTTGCATTACAACCATCCGGATTCATTGGACGTGATTCGCAATTTGGGAAATGAATTGGCAGCCGTTCTGGTGGAACCGGTACAAAGCCGCAGGCCGGATTTGCAGCCAGAATCATTTTTGAAAGAACTGCGGGCAATCACACAGCAATCCGGAACAGCTCTGATTATGGATGAAATTATTACAGGATTTCGGATCGGTCTCGGCGGCGCGCAGGAATGGTTCGACATCCAAGCAGATTTAGTCACTTACGGGAAAATCATCGGCGGCGGCCAGCCTCTAGGTATTGTTGCCGGAAAAGCAGAGTTCATGAATACGATCGATGGCGGCACATGGCAGTATGGAGACGACTCCTATCCAACGGATGAGGCAAAACGCACGTTTGTAGCGGGCACCTTTAATACTCACCCGCTTACGATGAGAATGTCATTAGCCGTGCTTCGATATCTTCAAGCCGAGGGAGAAACTCTGTATGAACGGTTAAATCAAAAGACAACCTACTTGGTTGATCAATTGAATTCCTATTTTGAACAATCGCAAGTGCCCATTCGTATGGTCCAATTTGGTTCCTTATTCCGGTTTGTCTCTTCGGTTGATAATGATTTGTTCTTTTACCATCTCAATTATAAAGGAGTCTATGTTTGGGAAGGACGCAACTGCTTCTTGTCTACGGCCCATACTTCCGATGATATTGCTTATATCATTCAAGCCGTTCAAGAAACGGTGAAAGATCTTCGCCGCGGCGGATTTATTCCAGAAGGGCCGGATTCTCCTAATGACGGAGGCCATAAAGAACCCGAAACATACGAGCTTTCTCCTGAACAAAAACAATTGGCTGTAGTATCCCAGTATGGGAATGATGCTTCTGCGGCATTGAATCAATCTATTATGCTAAAAGTGAAAGGGGCGGTGCAGCATACGCTGTTAAAACAAGCGGTGCGAAATATTGTAAAACGCCATGACGCTTTACGCACAGTCATTCATGTCGATGACGAAGTACAGCAAGTGCAGGCTCGAATAAATGTTGAAATTCCTATCATCGATTTTACCGGTTACCCGAATGAACAGCGAGAGTCGGAGGTTCAAAAATGGCTGACGGAAGATGCCAAGCGCCCGTTTCATTTCCATGAACAGAAGCCCTTGTTTAGAGTTCATGTACTTACGTCGAAACAAGACGAACATCTGATCGTTCTGACATTTCATCATATCATCGCCGATGGCTGGTCGATCGCTGTTTTTGTACAAGAGCTAGAGAGCACGTACGCCGCCATTGTACAAGGAAGCCCGCTTCCATCTCATGAGGTTGTTTCGTTTCGCCAATATTTAGATTGGCAGCAAGCTCAGATAGAGAATGGTCATTATGAAGAAGGAATTCGTTATTGGCGGCAGTATCTCTCTGAACCAATCCCGCAGGCAATCTTGACCAGTATGAGTTCTTCCCGTTATCCGCATGGTTACGAGGGAGATCGCTATACAGTTACACTGGACCGTCCATTGAGCAAGGCGATAAAGTCATTAAGCATTCGGATGAAAAATAGCGTTTTTGCAACTATTCTGGGAGCATTTCATCTTTTTCTGCAGCAGCTTACCAAGCAGGCTGGCCTTGTAATTGGGATTCCAACCGCAGGCCAGTTGCATATGAAACAACCTATGCTGGTTGGAAATTGTGTCAACATGGTTCCCGTGAAGAACACTGCTTCTTCAGAAAGCACATTAGCCGATTATCTGGGTCATATGAAGGAAAACATGGATCAAGTCATGCGGCATCAAGATGTTCCGATGACATTAGTGGCCAGCCAGCTTCCACACGATCAAATGCCGGATATGCGTATTATTTTTAATTTGGATAGACCTTTTCGAAAGCTGCATTTCGGACAGATGGAAGCTGAGCTCATTGCGTACCCTATAAAATGCATTTCATACGATTTATTTCTTAACGTAACGGAATTTGATCAAGAGTATGTTCTTGATTTCGATTTTAATACAAGCGTCATCAGTTCGGAAATCATGAACAAGTGGGGAACGGGCTTTGTAAACTTGCTGAAAAAAATGGTTGAGGGGGACTCCGCCTCTCTTGATTCCTTAAAAATGTTTTCGAAGGAAGATCAACACGACTTGCTTGAGCTGTATGCTGATCATCAGCTGCGAATCTCTTCAACATTAGACCATAAGGGTGTTCGTGCCGTTTACGAAGAGCCGGAAAATGAAACAGAGCTGCAAATTGCGCAGATTTGGGCGGAGCTTCTCGGCCTGGAGAAAGTGGGCAGATCTGACCACTTTCTGTCTCTGGGTGGAAACTCGCTAAAAGCGACGCTTATGCTTTCTAAGATTCAGCAAACATTTAATCAAAAGGTATCTATAGGGCAATTCTTCAGCCATCAGACTGTTAAGGAGTTGGCGAATTTCATCCGGGGTGAAAAGAATGTCAAGTATCCCCCGATGAAGCCTGTTGAGCAGAAAGCCTTTTACCGGACATCTCCAGCTCAGCAAAGAGTATATTTCCTGCATCAAATGGAACCGAATCAAGTTTCGCAAAATATGTTTGGCCAAATATCGATTATAGGGAAGTACGATGAAAAAGCCTTGATTGCATCCCTTCAACAGGTCATGCAGCGGCATGAAGCGTTTCGCACTTCTTTTCACATCATAGATGGTGAAATTGTGCAGCAGATTGCTGGCGAGCTTGATTTTAACGTTCGTGTCCATTCGATGGACCGTGAAGAATTTGAAGCCTACGCAGATGGGTATGTAAAACCTTTCCGTCTGGAACAAGCTCCTTTGGTTCGTGCGGAGCTGATCAAGGTCGATAACGAACAGGCTGAATTGCTCATCGATATGCATCATATCATTTCCGACGGCTATTCCATGAGCATACTTACAAATGAATTGTTCGCTTTGTATCATGGTAACCCATTACCGGAAATTCCATTTGAATATAAAGACTTCGCAGAGTGGCAAAACCAGCTGTTAATCGGAGAGGTCATGGAGCAGCAGGAAGAATACTGGCTCGAGCAATTCAAGCAAGAAGTTCCTATCCTTCAATTGCCGGCAGACGGTTCAAGAGCGATGGAATGGTCTTCCGAAGGGCAGCGTGTGACCTGTTCCTTGCAGTCGAGTTTAATCCGTTCGCTTCAAGAAATGGCGCAACAGAAGGGAACGACTCTGTATATGGTGCTTCTGGCTGCTTACAACGTGCTGCTTCACAAATATACGGGCCAAGAAGATATCGTCGTAGGCACGCCAGTTTCCGGAAGAAATCAACCGAATATTGAAAGCATGATTGGTATATTCATTCAAACCATGGGGATTCGCACGAAACCACAGGCTAATAAAAGGTTTACGGATTATTTGGACGAAGTTAAACGGCAAACGCTTGATGCGTTCGAAAACCAGGATTATCCGTTTGACTGGCTAGTAGAAAAAGTAAACGTACAACGGGAAACAACAGGTAAGTCACTATTTAACACAATGTTTGTGTATCAAAATATTGAATTTCAAGAGATCCATCAAGATGGGTGTACGTTTAGGGTAAAAGAACGTAATCCCGGAGTCTCTTTATATGATTTGATGTTAACGATCGAGGATGCAGAAAAACAGTTAGATATTCATTTCGATTTTAATCCAAACCAGTTTGAACAAGAAACGATTGAACAAATCATAAGGCACTACACCAGCCTTTTAGACAGTCTTGTTAAGGAGCCGGAGAAATCCTTGTCTTCCGTTCCTATGCTGTCTGACATCGAGAGGCACCAGCTTCTGATGGGGTGTAATGACACGGAGACGCCGTTTCCGCACAATGACACAGTATGTCAATGGTTTGAAACGCAAGCAGAACAGCGGCCTGATGATGAAGCCGTTATATTTGGCAATGAACGGTGCACGTACGGGCAGCTAAATGAGCGGGTAAATCAATTGGCGCGCACGTTAAGAACGAAGGGCGTTCAAGCGGATCAGTTTGTTGCCATCATCTGCCCGCATCGCATCGAGCTGATTGTTGGAATTTTGGCTGTTCTAAAAGCCGGCGGCGCATACGTGCCAATTGATCCGGAGTATCCAGAGGACCGGATACAATATATGCTGAAGGATTCAGAGGCTAAGATCGTTTTGGCACAGCTCGATTTGCATAAACACTTAACGTTTGATGCTGACGTTGTGCTTTTGGATGAGGAAAGCTCATATCATGAGGATCGTTCGAATCTTGAACCGACCTGCGGTGCAAATGATTTGGCATACATGATCTATACGTCGGGCTCCACAGGGAACCCGAAAGGTGTACTCATTGAGCACCGGGGATTAGCTAATTATATTGAGTGGGCGAAAGAGGTTTATGTGAATGATGAGAAAACCAACTTCCCTTTATACTCGTCCATCTCTTTTGATCTAACGGTGACGTCGATTTTTACACCGCTGGTTACAGGAAATACCATCATTGTCTTTGATGGTGAAGACAAAAGTGCGGTGCTTTCAACAATTATGCAGGATCCGAGAATAGATATCATCAAATTGACGCCGGCGCATTTGCATGTGCTCAAAGAAATGAAGATAGCAGATGGAACGACAATTCGAAAAATGATTGTCGGCGGGGAAAATTTAAGCACCCGGCTTGCCCAAAGTGTCAGTGAGCAGTTTAAAGGCCAACTGGACATATTCAATGAATACGGACCGACAGAAGCGGTCGTCGGATGTATGATTTATCGGTACGACACTAAACGTGACAGGCGAGAATTTGTGCCAATAGGCTCCCCTGCCGCCAATACGAGCATTTATGTGTTGGATGCCAGCATGAACTTGGTTCCGGTCGGCGTACCGGGTGAAATGTATATCGGTGGAGCCGGTGTAGCCAGAGGATACTGGAATCGCCCGGATTTAACAGCAGAGAAGTTCGTTCACAACCCGTTTGCTCCGGGAACGATAATGTACAAAACGGGTGACTTGGCAAAACGATTACGTGATGGAAATCTCATATATTTAGGCCGAATCGATGAACAAGTCAAAATCCGAGGACATCGAATTGAACTTGGTGAAGTTGAAGCTGCAATGCATAAAGTGGAAGCGGTCCAAAAGGCCGTAGTTTTAGCCAGAGAAGAAGAGGATGGCTTACAACAACTGTGTGCGTATTATGTGAGCAATAAACCTATAACAATTGCGGAGATTAGAGAACAATTATCACTGGAGCTGCCGGACTACATGGTTCCGTCCCATTATATCCAACTTGAGCAATTACCGTTAACGTCCAACGGGAAAATAAATCGTAAAGCACTGCCTGCACCAGAGGTAAGTTTAGAGCAAATAGCTGAATATGTACCGCCAGGCAATGAGGTTGAATCTAAGCTTGCAGTCTTATGGCAAGAGATGCTCGGAATACATCGTGTGGGGATCAAGCACAATTTCTTCGATCTTGGAGGAAATTCCATACGCGCGACGGCCTTAGCCGCCAGAATCCACAAAGAACTGGATGTCAATCTGTCTGTAAAAGACATATTTAAGTTTCCTACTATTGAACAGTTGGCTAACATGGCGTTACGCATGGAGAAAATTCGATATGTATCAATTCCGTCTGCACAGAAAATCTCCTATTATCCAGTTTCTTCGGCACAGAAACGGATGTATTTGTTAAGTCATACAGAAGGAGGCGAGCTGACGTACAATATGACGGGCGCCATGAGTGTAGAAGGGGCTATTGATCTAGAACGATTGACCGCTGCTTTTCAAAAATTAATTGAACGTCATGAAGTTTTGCGGACCAGCTTTGAACTATACGAAGGCGAGCCGGCACAGCGAATTCATCCAAGCATTGAATTTACAATAGAACAGATTCAAGCGAGAGAAGAGGAAGTGGAAGACCATGTACTTGATTTTATCAAATCGTTTGATTTAGCCAAGCCGCCGTTAATGCGAGTGGGACTGATTGAACTTACACCCGAAAAGCATGTACTGCTAGTCGATATGCATCATATCATTTCCGATGGCGTGTCTATGAACATTCTAATGAAAGATTTAAATCAATTTTATAAAGGGATCGAACCGGATCCGCTTCCCATTCAATATAAGGACTATGCGGTTTGGCAGCAAACGGAAGCTCAGAGGCAAAACATCAAAAAACAGGAAGCGTATTGGCTTAATCGTTTTCATGATGAGATTCCTGTATTGGATATGCCAACGGATTACGAGAGACCTGCTATACGCGATTACGAAGGCGAATCATTTGAATTTCTTATACCGATAGAATTAAAACAGCGCTTAAGTCAAATGGAAGAAGCTACAGGAACAACATTGTATATGATTTTAATGGCAGCTTATACAATTCTTTTATCCAAATACAGCGGACAGGAAGATATCGTCGTAGGGACCCCGGTCTCCGGCCGAAGTCATATGGATGTAGAGTCTGTTGTGGGAATGTTTGTAAACACCTTAGTCATTCGCAATCACCCGGCAGGCCGTAAAATATTCGAGGATTACTTAAACGAAGTGAAGGAAAACATGCTAAATGCCTATCAAAATCAAGACTATCCATTGGAAGAATTGATCCAACATGTACATCTTCTAAAAGATTCAAGCCGCAACCCTTTATTCGATACGATGTTTGTGCTGCAAAATCTCGATCAGGTTGAATTGAACCTTGATTCCCTTCGATTCACGCCTTATAAGCTTCATCATACAGTTGCCAAATTCGATTTGACCTTGTCGATTCAGACAGATCAAGACAAACATCACGGTCTGTTCGAATATTCGAAGAAACTATTTAAGAAAAGCAGAATCGAAGCTTTGTCAAAAGACTATTTACACATCTTATCCGTTATCAGTCAACAGCCAAGTATACAAATCGAACATATCGAATTAAGCGGCAGCACCGCGGAAGATGATAACTTGATCCATTCTATTGAACTGAACTTTTAA SEQ ID NO: 10 Amino AcidMycosubtilin synthase subunit A MycA (YP_003866245.1)Interior Acyl Binding Pocket domain underlined 1MYTSQFQTLV DVIRNRSNIS DRGIRFIESD KIETFVSYRQ LFDEAQGFLG YLQHIGIQPK 61QEIVFQIQEN KSFVVAFWAC LLGGMIPVPV SIGEDNDHKL KVWRIWNILN NPFLLASETV 121LDKMKKFAAD HDLQDFHHQL IEKSDIIQDR IYDHPASQYE PEADELAFIQ FSSGSTGDPK 181GVMLTHHNLI HNTCAIRNAL AIDLKDTLLS WMPLTHDMGL IACHLVPALA GINQNLMPTE 241LFIRRPILWM KKAHEHKASI LSSPNFGYNY FLKFLKDNKS YDWDLSHIRV IANGAEPILP 301ELCDEFLTRC AAFNMKRSAI LNVYGLAEAS VGATFSNIGE RFVPVYLHRD HLNLGERAVE 361VSKEDQNCAS FVEVGKPIDY CQIRICNEAN EGLEDGFIGH IQIKGENVTQ GYYNNPESTN 421RALTPDGWVK TGDLGFIRKG NLVVTGREKD IIFVNGKNVY PHDIERVAIE LEDIDLGRVA 481ACGVYDQETR SREIVLFAVY KKSAEQFAPL VKDIKKHLYQ RGGWSIKEIL PIRKLPKTTS 541GKVKRYELAE QYESGKFALE STKIKEFLEG HSTEPVQTPI HEIETALLSI FSEVMDGKKI 601HLNDHYFDMG ATSLQLSQIA ERIEQKFGCE LTVADLFTYP SIADLAAFLV ENHSEIKQTD 661TAKPSRSSSK DIAIIGMSLN VPGASNKSDF WHLLENGEHG IREYPAPRVK DAIDYLRSIK 721SERNEKQFVK GGYLDEIDRF DYSFFGLAPK TAKFMDPNQR LFLQSAWHAI EDAGYAGDTI 781SGSQLGVYVG YSKVGYDYER LLSANYPEEL HHYIVGNLPS VLASRIAYFL NLKGPAVTVD 841TACSSSLVAV HMACKALLTG DCEMALAGGI RTSLLPMRIG LDMESSDGLT KTFSKDSDGT 901GSGEGVAAVL LKPLQAAIRD GDHIYGVIKG SAINQDGTTV GITAPSPAAQ TEVIEMAWKD 961AGIAPETLSF IEAHGTGTKL GDPVEFNGLC KAFEKVTEKK QFCAIGSVKA NIGHLFEAAG 1021IVGLIKSALM LNHKKIPPLA HFNKPNPLIP FHSSPFYVNQ EVMDFTPEDR PLRGGISSFG 1081FSGTNAHVVL EEYTPESEYA PEDGNDPHLF VLSAHTEASL YELTHQYRQY ISDDSQSSLR 1141SICYTASTGR AHLDYCLAMI VSSNQELIDK LTSLIQGERN LPQVHFGYKN IKEMQPAEKD 1201NLSKQISDLM QHRPCTKDER ITWLNRIAEL YVQRAVIDWR AVYSNEVVQK TPLPLYPFER 1261NRCWVEAVYE SAKERKEKGE VALDINHTKT HIESFLKTVI SNASGIRADE IDSNAHFIGF 1321GLDSIMLTQV KKAIADEFNV DIPMERFFDT MNNIESVVDY LAENVPSAAS TPPQESVTAQ 1381EELVISGAQP ELEHQEHMLD KIIASQNQLI QQTLQAQLDS FNLLRNNSHF VSKESEISQD 1441KTSLSPKSVT AKKNSAQEAK PYIPFQRQTL NEQVNYTPQQ RQYLESFIEK YVDKTKGSKQ 1501YTDETRFAHA NNRNLSSFRS YWKEMVYPII AERSDGSRMW DIDGNEYIDI TMGFGVNLFG 1561HHPSFITQTV VDSTHSALPP LGPMSNVAGE VADRIRACTG VERVAFYNSG TEAVMVALRL 1621ARAATGRTKV VVFAGSYHGT FDGVLGVANT KGGAEPANPL APGIPQSFMN DLIILHYNHP 1681DSLDVIRNLG NELAAVLVEP VQSRRPDLQP ESFLKELRAI TQQSGTALIM DEIITGFRIG 1741LGGAQEWFDI QADLVTYGKI IGGGQPLGIV AGKAEFMNTI DGGTWQYGDD SYPTDEAKRT 1801FVAGTFNTHP LTMRMSLAVL RYLQAEGETL YERLNQKTTY LVDQLNSYFE QSQVPIRMVQ 1861FGSLFRFVSS VDNDLFFYHL NYKGVYVWEG RNCFLSTAHT SDDIAYIIQA VQETVKDLRR 1921GGFIPEGPDS PNDGGHKEPE TYELSPEQKQ LAVVSQYGND ASAALNQSIM LKVKGAVQHT 1981LLKQAVRNIV KRHDALRTVI HVDDEVQQVQ ARINVEIPII DFTGYPNEQR ESEVQKWLTE 2041DAKRPFHFHE QKPLFRVHVL TSKQDEHLIV LTFHHIIADG WSIAVFVQEL ESTYAAIVQG 2101SPLPSHEVVS FRQYLDWQQA QIENGHYEEG IRYWRQYLSE PIPQAILTSM SSSRYPHGYE 2161GDRYTVTLDR PLSKAIKSLS IRMKNSVFAT ILGAFHLFLQ QLTKQAGLVI GIPTAGQLHM 2221KQPMLVGNCV NMVPVKNTAS SESTLADYLG HMKENMDQVM RHQDVPMTLV ASQLPHDQMP 2281DMRIIFNLDR PFRKLHFGQM EAELIAYPIK CISYDLFLNV TEFDQEYVLD FDFNTSVISS 2341EIMNKWGTGF VNLLKKMVEG DSASLDSLKM FSKEDQHDLL ELYADHQLRI SSTLDHKGVR 2401AVYEEPENET ELQIAQIWAE LLGLEKVGRS DHFLSLGGNS LKATLMLSKI QQTFNQKVSI 2461GQFFSHQTVK ELANFIRGEK NVKYPPMKPV EQKAFYRTSP AQQRVYFLHQ MEPNQVSQNM 2521FGQISIIGKY DEKALIASLQ QVMQRHEAFR TSFHIIDGEI VQQIAGELDF NVRVHSMDRE 2581EFEAYADGYV KPFRLEQAPL VRAELIKVDN EQAELLIDMH HIISDGYSMS ILTNELFALY 2641HGNPLPEIPF EYKDFAEWQN QLLIGEVMEQ QEEYWLEQFK QEVPILQLPA DGSRAMEWSS 2701EGQRVTCSLQ SSLIRSLQEM AQQKGTTLYM VLLAAYNVLL HKYTGQEDIV VGTPVSGRNQ 2761PNIESMIGIF IQTMGIRTKP QANKRFTDYL DEVKRQTLDA FENQDYPFDW LVEKVNVQRE 2821TTGKSLFNTM FVYQNIEFQE IHQDGCTFRV KERNPGVSLY DLMLTIEDAE KQLDIHFDFN 2881PNQFEQETIE QIIRHYTSLL DSLVKEPEKS LSSVPMLSDI ERHQLLMGCN DTETPFPHND 2941TVCQWFETQA EQRPDDEAVI FGNERCTYGQ LNERVNQLAR TLRTKGVQAD QFVAIICPHR 3001IELIVGILAV LKAGGAYVPI DPEYPEDRIQ YMLKDSEAKI VLAQLDLHKH LTFDADVVLL 3061DEESSYHEDR SNLEPTCGAN DLAYMIYTSG STGNPKGVLI EHRGLANYIE WAKEVYVNDE 3121KTNFPLYSSI SFDLTVTSIF TPLVTGNTII VFDGEDKSAV LSTIMQDPRI DIIKLTPAHL 3181HVLKEMKIAD GTTIRKMIVG GENLSTRLAQ SVSEQFKGQL DIFNEYGPTE AVVGCMIYRY 3241DTKRDRREFV PIGSPAANTS IYVLDASMNL VPVGVPGEMY IGGAGVARGY WNRPDLTAEK 3301FVHNPFAPGT IMYKTGDLAK RLRDGNLIYL GRIDEQVKIR GHRIELGEVE AAMHKVEAVQ 3361KAVVLAREEE DGLQQLCAYY VSNKPITIAE IREQLSLELP DYMVPSHYIQ LEQLPLTSNG 3421KINRKALPAP EVSLEQIAEY VPPGNEVESK LAVLWQEMLG IHRVGIKHNF FDLGGNSIRA 3481TALAARIHKE LDVNLSVKDI FKFPTIEQLA NMALRMEKIR YVSIPSAQKI SYYPVSSAQK 3541RMYLLSHTEG GELTYNMTGA MSVEGAIDLE RLTAAFQKLI ERHEVLRTSF ELYEGEPAQR 3601IHPSIEFTIE QIQAREEEVE DHVLDFIKSF DLAKPPLMRV GLIELTPEKH VLLVDMHHII 3661SDGVSMNILM KDLNQFYKGI EPDPLPIQYK DYAVWQQTEA QRQNIKKQEA YWLNRFHDEI 3721PVLDMPTDYE RPAIRDYEGE SFEFLIPIEL KQRLSQMEEA TGTTLYMILM AAYTILLSKY 3781SGQEDIVVGT PVSGRSHMDV ESVVGMFVNT LVIRNHPAGR KIFEDYLNEV KENMLNAYQN 3841QDYPLEELIQ HVHLLKDSSR NPLFDTMFVL QNLDQVELNL DSLRFTPYKL HHTVAKFDLT 3901LSIQTDQDKH HGLFEYSKKL FKKSRIEALS KDYLHILSVI SQQPSIQIEH IELSGSTAED 3961DNLIHSIELN F SEQ ID NO: 11 NucleotideInterior Acyl Binding Pocket of mycAATCCGGAATGCGCTGGCTATCGACTTAAAAGATACTCTTTTATCTTGGATGCCCTTAACCCATGACATGGGGCTCATAGCTTGCCACCTTGTTCCTGCCTTAGCCGGAATCAATCAAAATTTAATGCCGACAGAATTATTTATTCGAAGACCTATTCTCTGGATGAAAAAAGCTCATGAACATAAAGCCAGCATTCTATCCTCACCTAATTTTGGATACAATTACTTTCTTAAATTTCTGAAAGACAATAAAAGTTACGACTGGGATTTATCCCATATCAGGGTCATTGCAAACSEQ ID NO: 12 Amino Acid Interior Acyl Binding Pocket of MycAIRNALAIDLKDTLLSWMPLTHDMGLIACHLVPALAGINQNLMPTELFIRRPILWMKKAHEHKASILSSPNFGYNYFLKFLKDNKSYDWDLSHIRVIAN SEQ ID NO: 13 Nucleotide dptEInterior Acyl Binding Pocket domain underlinedACCESSION: AY787762 REGION: 49421 . . . 51214VERSION: AY787762.1 GI: 60650890 1gtgagtgaga gccgctgtgc cgggcagggc ctggtggggg cactgcggac ctgggcacgg 61acacgtgccc gggagactgc cgtggttctc gtacgggaca ccggaaccac cgacgacacg 121gcgtcggtgg actacggaca gctggacgag tgggccagaa gcatcgcggt gaccctccga 181cagcaactcg cgccgggggg acgggcactt ctgctgctgc cgtccggccc ggagttcacg 241gccgcgtacc tcggctgcct gtacgcgggt ctggccgccg taccggcgcc gctgcccggg 301gggcgccact tcgaacgccg ccgtgtcgcg gccatcgccg ccgacagcgg agccggcgtg 361gtgctgaccg tcgcgggtga gaccgcctcc gtccacgact ggctgaccga gaccacggcc 421ccggctactc gcgtcgtggc cgtggacgac cgggcggcgc tcggcgaccc ggcgcagtgg 481gacgacccgg gcgtcgcgcc cgacgacgtg gctctcatcc agtacacctc gggctcgacc 541ggcaacccca agggcgtggt cgtgacccac gccaacctgc tggcgaacgc gcggaat ctc 601gccgaggcct gcgagctgac cgccgccact cccatgggcg gctggctgcc catgtaccac 661gacatggggc tcctgggcac gctgacaccg gccctgtacc tcggcaccac gtgcgtgctg 721atgagctcca cggcattcat caaacggccg cacctgtggc tacggaccat cgaccggttc 781ggcctggtct ggtcgtcggc tcccgacttc gcgtacgaca tgtgtctgaa gcgcgtcacc 841gacgagcaga tcgccgggct ggacctgtcc cgctggcggt gggccggcaa c ggcgcggag 901cccatccggg cagccaccgt acgggccttc ggcgaacggt tcgcccggta cggcctgcgc 961cccgaggcgc tcaccgccgg ctacgggctg gccgaggcca ccctgttcgt gtcgaggtcg 1021caggggctgc acacggcacg agtcgccacc gccgccctcg aacgccacga attccgcctc 1081gccgtacccg gcgaggcagc ccgggagatc gtcagctgcg gtcccgtcgg ccacttccgc 1141gcccgcatcg tcgaacccgg cgggcaccgt gttctgccgc ccggccaggt cggcgagctg 1201gtcctccagg gagccgccgt ctgcgccggc tactggcagg ccaaggagga gaccgagcag 1261accttcggcc tcaccctcga cggcgaggac ggtcactggc tgcgcaccgg cgatctcgcc 1321gccctgcacg aagggaatct ccacatcacc ggccgctgca aagaggccct ggtgatacga 1381ggacgcaatc tgtacccgca ggacatcgag cacgaactcc gcctgcaaca cccggaactt 1441gagagcgtcg gcgccgcgtt caccgtcccg gcggcacctg gcacgccggg cttgatggtg 1501gtccacgaag tccgcacccc ggtccccgcc gacgaccacc cggccctggt cagcgccctg 1561cgggggacga tcaaccgcga attcggactc gacgcccagg gcatcgccct ggtgagccgc 1621ggcaccgtac tgcgtaccac cagcggcaag gtccgccggg gcgccatgcg tgacctctgc 1681ctccgcgggg agctgaacat cgtccacgcg gacaagggct ggcacgccat cgccggcacg 1741gccggagagg acatcgcccc cactgaccac gctccacatc cgcaccccgc gtaaSEQ ID NO: 14 Amino Acid DptE (AAX31555.1)Interior Acyl Binding Pocket domain underlined 1MSESRCAGQG LVGALRTWAR TRARETAVVL VRDTGTTDDT ASVDYGQLDE WARSIAVTLR 61QQLAPGGRAL LLLPSGPEFT AAYLGCLYAG LAAVPAPLPG GRHFERRRVA AIAADSGAGV 121VLTVAGETAS VHDWLTETTA PATRVVAVDD RAALGDPAQW DDPGVAPDDV ALIQYTSGST 181GNPKGVVVTH ANLLANARNL AEACELTAAT PMGGWLPMYH DMGLLGTLTP ALYLGTTCVL 241MSSTAFIKRP HLWLRTIDRF GLVWSSAPDF AYDMCLKRVT DEQIAGLDLS RWRWAGNGAE 301PIRAATVRAF GERFARYGLR PEALTAGYGL AEATLFVSRS QGLHTARVAT AALERHEFRL 361AVPGEAAREI VSCGPVGHFR ARIVEPGGHR VLPPGQVGEL VLQGAAVCAG YWQAKEETEQ 421TFGLTLDGED GHWLRTGDLA ALHEGNLHIT GRCKEALVIR GRNLYPQDIE HELRLQHPEL 481ESVGAAFTVP AAPGTPGLMV VHEVRTPVPA DDHPALVSAL RGTINREFGL DAQGIALVSR 541GTVLRTTSGK VRRGAMRDLC LRGELNIVHA DKGWHAIAGT AGEDIAPTDH APHPHPASEQ ID NO: 15 Nucleotide Interior Acyl Binding Pocket of dptECTCGCCGAGGCCTGCGAGCTGACCGCCGCCACTCCCATGGGCGGCTGGCTGCCCATGTACCACGACATGGGGCTCCTGGGCACGCTGACACCGGCCCTGTACCTCGGCACCACGTGCGTGCTGATGAGCTCCACGGCATTCATCAAACGGCCGCACCTGTGGCTACGGACCATCGACCGGTTCGGCCTGGTCTGGTCGTCGGCTCCCGACTTCGCGTACGACATGTGTCTGAAGCGCGTCACCGACGAGCAGATCGCCGGGCTGGACCTGTCCCGCTGGCGGTGGGCCGGCAACSEQ ID NO: 16 Amino Acid Interior Acyl Binding Pocket of DptELAEACELTAATPMGGWLPMYHDMGLLGTLTPALYLGTTCVLMSSTAFIKRPHLWLRTIDRFGLVWSSAPDFAYDMCLKRVTDEQIAGLDLSRWRWAGN SEQ ID NO: 17 NucleotideEngineered NonA with safB Interior Acyl Binding Pocket domain (underlined)ATGGTTGGTCAATTTGCAAATTTCGTCGATCTGCTCCAGTACAGAGCTAAACTTCAGGCGCGGAAAACCGTGTTTAGTTTTCTGGCTGATGGCGAAGCGGAATCTGCGGCCCTGACCTACGGAGAATTAGACCAAAAAGCCCAGGCGATCGCCGCTTTTTTGCAAGCTAACCAGGCTCAAGGGCAACGGGCATTATTACTTTATCCACCGGGTTTAGAGTTTATCGGTGCCTTTTTGGGATGTTTGTATGCTGGTGTTGTTGCGGTGCCAGCTTACCCACCACGGCCGAATAAATCCTTTGACCGCCTCCATAGCATTATCCAAGATGCCCAGGCAAAATTTGCCCTCACCACAACAGAACTTAAAGATAAAATTGCCGATCGCCTCGAAGCTTTAGAAGGTACGGATTTTCATTGTTTGGCTACAGATCAAGTTGAATTAATTTCAGGAAAAAATTGGCAAAAACCGAACATTTCCGGCACAGATCTCGCTTTTTTGCAATACACCAGTGGCTCCACGGGCGATCCTAAAGGAGTGATGGTTTCCCACCACAATTTGATCCACAACTCCGGCTTGATTTTTACCTCTTTTCATATGAATGATGAAACCATTATTTTCAGCTGGCTGCCCCCACATCATGATATGGGTTTGATTGGCTGCATTCTGACCCCCATCTATGGTGGAATTCAGGCAATCATGATGTCCCCTTTCTCATTTTTACAAAACCCGCTTTCCTGGTTAAAACATATTACCAAATACAAAGCAACTATCAGTGGAAGCCCTAACTTCGCTTACGATTATTGTGTCAAACGAATCAGGGAAGAAAAAAAAGAAGGGCTGGATTTAAGTTCATGGGTGACTGCTTTCAACGGGGCCGAACCGATCCGCGCTGTGACCCTCGAAAATTTTGCGAAAACCTTCGCTACAGCAGGCTTTCAAAAATCAGCATTTTATCCCTGTTATGGTATGGCTGAAACCACCCTGATCGTTTCCGGTGGTAATGGTCGTGCCCAGCTTCCCCAGGAAATTATCGTCAGCAAACAGGGCATCGAAGCAAACCAAGTTCGCCCTGCCCAAGGGACAGAAACAACGGTGACCTTGGTCGGCAGTGGTGAAGTGATTGGCGACCAAATTGTCAAAATTGTTGACCCCCAGGCTTTAACAGAATGTACCGTCGGTGAAATTGGCGAAGTATGGGTTAAGGGCGAAAGTGTTGCCCAGGGCTATTGGCAAAAGCCAGACCTCACCCAGCAACAATTCCAGGGAAACGTCGGTGCAGAAACGGGCTTTTTACGCACGGGCGATCTGGGTTTTTTGCAAGGTGGCGAACTGTATATTACGGGTCGTTTAAAGGATCTCCTGATTATCCGGGGGCGCAACCACTATCCCCAGGACATTGAATTAACCGTCGAAGTGGCCCATCCCGCTTTACGACAGGGGGCCGGAGCCGCTGTATCAGTAGACGTTAACGGGGAAGAACAGTTAGTCATTGTCCAGGAAGTTGAGCGTAAATATGCCCGCAAATTAAATGTCGCGGCAGTAGCCCAAGCTATTCGTGGGGCGATCGCCGCCGAACATCAACTGCAACCCCAGGCCATTTGTTTTATTAAACCCGGTAGCATTCCCAAAACATCCAGCGGGAAGATTCGTCGCCATGCCTGCAAAGCTGGTTTTCTAGACGGAAGCTTGGCTGTGGTTGGGGAGTGGCAACCCAGCCACCAAAAAGAAGGAAAAGGAATTGGGACACAAGCCGTTACCCCTTCTACGACAACATCAACGAATTTTCCCCTGCCTGACCAGCACCAACAGCAAATTGAAGCCTGGCTTAAGGATAATATTGCCCATCGCCTCGGCATTACGCCCCAACAATTAGACGAAACGGAACCCTTTGCAAGTTATGGGCTGGATTCAGTGCAAGCAGTACAGGTCACAGCCGACTTAGAGGATTGGCTAGGTCGAAAATTAGACCCCACTCTGGCCTACGATTATCCGACCATTCGCACCCTGGCTCAGTTTTTGGTCCAGGGTAATCAAGCGCTAGAGAAAATACCACAGGTGCCGAAAATTCAGGGCAAAGAAATTGCCGTGGTGGGTCTCAGTTGTCGTTTTCCCCAAGCTGACAACCCCGAAGCTTTTTGGGAATTATTACGTAATGGTAAAGATGGAGTTCGCCCCCTTAAAACTCGCTGGGCCACGGGAGAATGGGGTGGTTTTTTAGAAGATATTGACCAGTTTGAGCCGCAATTTTTTGGCATTTCCCCCCGGGAAGCGGAACAAATGGATCCCCAGCAACGCTTACTGTTAGAAGTAACCTGGGAAGCCTTGGAACGGGCAAATATTCCGGCAGAAAGTTTACGCCATTCCCAAACGGGGGTTTTTGTCGGCATTAGTAATAGTGATTATGCCCAGTTGCAGGTGCGGGAAAACAATCCGATCAATCCCTACATGGGGACGGGCAACGCCCACAGTATTGCTGCGAATCGTCTGTCTTATTTCCTCGATCTCCGGGGCGTTTCTCTGAGCATCGATACGGCCTGTTCCTCTTCTCTGGTGGCGGTACATCTGGCCTGTCAAAGTTTAATCAACGGCGAATCGGAGTTGGCGATCGCCGCCGGGGTGAATTTGATTTTGACCCCCGATGTGACCCAGACTTTTACCCAGGCGGGCATGATGAGTAAGACGGGCCGTTGCCAGACCTTTGATGCCGAGGCTGATGGCTATGTGCGGGGCGAAGGTTGTGGGGTCGTTCTCCTCAAACCCCTGGCCCAGGCAGAACGGGACGGGGATAATATTCTCGCGGTGATCCACGGTTCGGCGGTGAATCAAGATGGACGCAGTAACGGTTTGACGGCTCCCAACGGGCGATCGCAACAGGCCGTTATTCGCCAAGCCCTGGCCCAAGCCGGCATTACCGCCGCCGATTTAGCTTACCTAGAGGCCCACGGCACCGGCACGCCCCTGGGTGATCCCATTGAAATTAATTCCCTGAAGGCGGTTTTACAAACGGCGCAGCGGGAACAGCCCTGTGTGGTGGGTTCTGTGAAAACAAACATTGGTCACCTCGAGGCAGCGGCGGGCATCGCGGGCTTAATCAAGGTGATTTTGTCCCTAGAGCATGGAATGATTCCCCAACATTTGCATTTTAAGCAGCTCAATCCCCGCATTGATCTAGACGGTTTAGTGACCATTGCGAGCAAAGATCAGCCTTGGTCAGGCGGGTCACAAAAACGGTTTGCTGGGGTAAGTTCCTTTGGGTTTGGTGGCACCAATGCCCACGTGATTGTCGGGGACTATGCTCAACAAAAATCTCCCCTTGCTCCTCCGGCTACCCAAGACCGCCCTTGGCATTTGCTGACCCTTTCTGCTAAAAATGCCCAGGCCTTAAATGCCCTGCAAAAAAGCTATGGAGACTATCTGGCCCAACATCCCAGCGTTGACCCACGCGATCTCTGTTTGTCTGCCAATACCGGGCGATCGCCCCTCAAAGAACGTCGTTTTTTTGTCTTTAAACAAGTCGCCGATTTACAACAAACTCTCAATCAAGATTTTCTGGCCCAACCACGCCTCAGTTCCCCCGCAAAAATTGCCTTTTTGTTTACGGGGCAAGGTTCCCAATACTACGGCATGGGGCAACAACTGTACCAAACCAGCCCAGTATTTCGGCAAGTGCTGGATGAGTGCGATCGCCTCTGGCAGACCTATTCCCCCGAAGCCCCTGCCCTCACCGACCTGCTGTACGGTAACCATAACCCTGACCTCGTCCACGAAACTGTCTATACCCAGCCCCTCCTCTTTGCTGTTGAATATGCGATCGCCCAACTATGGTTAAGCTGGGGCGTGACGCCAGACTTTTGCATGGGCCATAGCGTCGGCGAATATGTCGCGGCTTGTCTGGCGGGGGTATTTTCCCTGGCAGACGGCATGAAATTAATTACGGCCCGGGGCAAACTGATGCACGCCCTACCCAGCAATGGCAGTATGGCGGCGGTCTTTGCCGATAAAACGGTCATCAAACCCTACCTATCGGAGCATTTGACCGTCGGAGCCGAAAACGGTTCCCATTTGGTGCTATCAGGAAAGACCCCCTGCCTCGAAGCCAGTATTCACAAACTCCAAAGCCAAGGGATCAAAACCAAACCCCTCAAGGTTTCCCATGCTTTCCACTCCCCTTTGATGGCTCCCATGCTGGCAGAGTTTCGGGAAATTGCTGAACAAATTACTTTCCACCCGCCGCGTATCCCGCTCATTTCCAATGTCACGGGCGGCCAGATTGAAGCGGAAATTGCCCAGGCCGACTATTGGGTTAAGCACGTTTCGCAACCCGTCAAATTTGTCCAGAGCATCCAAACCCTGGCCCAAGCGGGTGTCAATGTTTATCTCGAAATCGGCGTAAAACCAGTGCTCCTGAGTATGGGACGCCATTGCTTAGCTGAACAAGAAGCGGTTTGGTTGCCCAGTTTACGTCCCCATAGTGAGCCTTGGCCGGAAATTTTGACCAGTCTCGGCAAACTGTATGAGCAAGGGCTAAACATTGACTGGCAGACCGTGGAAGCTGGCGATCGCCGCCGGAAACTGATTCTGCCCACCTATCCCTTCCAACGGCAACGATATTGGTTTAATCAAGGCTCTTGGCAAACTGTTGAGACCGAATCTGTGAACCCAGGCCCTGACGATCTCAATGATTGGTTGTATCAGGTGGCGTGGACGCCCCTGGACACTTTGCCCCCGGCCCCTGAACCGTCGGCTAAGCTGTGGTTAATCTTGGGCGATCGCCATGATCACCAGCCCATTGAAGCCCAATTTAAAAACGCCCAGCGGGTGTATCTCGGCCAAAGCAATCATTTTCCGACGAATGCCCCCTGGGAAGTATCTGCCGATGCGTTGGATAATTTATTTACTCACGTCGGCTCCCAAAATTTAGCAGGCATCCTTTACCTGTGTCCCCCAGGGGAAGACCCAGAAGACCTAGATGAAATTCAAAAGCAAACCAGTGGCTTCGCCCTCCAACTGATCCAAACCCTGTATCAACAAAAGATCGCGGTTCCCTGCTGGTTTGTGACCCACCAGAGCCAACGGGTGCTTGAAACCGATGCTGTCACCGGATTTGCCCAAGGGGGATTATGGGGACTCGCCCAGGCGATCGCCCTCGAACATCCAGAGTTGTGGGGGGGAATTATTGATGTCGATGACAGCCTGCCAAATTTTGCCCAGATTTGCCAACAAAGACAGGTGCAGCAGTTGGCCGTGCGGCACCAAAAACTCTACGGGGCACAGCTCAAAAAGCAACCGTCACTGCCCCAGAAAAATCTCCAGATTCAACCCCAACAGACCTATCTAGTGACAGGGGGACTGGGGGCCATTGGCCGTAAAATTGCCCAATGGCTAGCCGCAGCAGGAGCAGAAAAAGTAATTCTCGTCAGCCGGCGCGCTCCGGCAGCGGATCAGCAGACGTTACCGACCAATGCGGTGGTTTATCCTTGCGATTTAGCCGACGCAGCCCAGGTGGCAAAGCTGTTTCAAACCTATCCCCACATCAAAGGAATTTTCCATGCGGCGGGTACCTTAGCTGATGGTTTGCTGCAACAACAAACTTGGCAAAAGTTCCAGACCGTCGCCGCCGCCAAAATGAAAGGGACATGGCATCTGCACCGCCATAGTCAAAAGCTCGATCTGGATTTTTTTGTGTTGTTTTCCTCTGTGGCAGGGGTGCTCGGTTCACCGGGACAGGGGAATTATGCCGCCGCAAACCGGGGCATGGCGGCGATCGCCCAATATCGACAAGCCCAAGGTTTACCCGCCCTGGCGATCCATTGGGGGCCTTGGGCCGAAGGGGGAATGGCCAACTCCCTCAGCAACCAAAATTTAGCGTGGCTGCCGCCCCCCCAGGGACTAACAATCCTCGAAAAAGTCTTGGGCGCCCAGGGGGAAATGGGGGTCTTTAAACCGGACTGGCAAAACCTGGCCAAACAGTTCCCCGAATTTGCCAAAACCCATTACTTTGCAGCCGTTATTCCCTCTGCTGAGGCTGTGCCCCCAACGGCTTCAATTTTTGACAAATTAATCAACCTAGAAGCTTCTCAGCGGGCTGACTATCTACTGGATTATCTGCGGCGGTCTGTGGCGCAAATCCTCAAGTTAGAAATTGAGCAAATTCAAAGCCACGATAGCCTGTTGGATCTGGGCATGGATTCGTTGATGATCATGGAGGCGATCGCCAGCCTCAAGCAGGATTTACAACTGATGTTGTACCCCAGGGAAATCTACGAACGGCCCAGACTTGATGTGTTGACGGCCTATCTAGCGGCGGAATTCACCAAGGCCCATGATTCTGAAGCAGCAACGGCGGCAGCAGCGATTCCCTCCCAAAGCCTTTCGGTCAAAACAAAAAAACAGTGGCAAAAACCTGACCACAAAAACCCGAATCCCATTGCCTTTATCCTCTCTAGCCCCCGGTCGGGTTCGACGTTGCTGCGGGTGATGTTAGCCGGACATCCGGGGTTATATTCGCCGCCAGAGCTGCATTTGCTCCCCTTTGAGACTATGGGCGATCGCCACCAGGAATTGGGTCTATCCCACCTCGGCGAAGGGTTACAACGGGCCTTAATGGATCTAGAAAACCTCACCCCAGAGGCAAGCCAGGCGAAGGTCAACCAATGGGTCAAAGCGAATACACCCATTGCAGACATCTATGCCTATCTCCAACGGCAGGCGGAACAACGTTTACTCATCGACAAATCTCCCAGCTACGGCAGCGATCGCCATATTCTAGACCACAGCGAAATCCTCTTTGACCAGGCCAAATATATCCATCTGGTACGCCATCCCTACGCGGTGATTGAATCCTTTACCCGACTGCGGATGGATAAACTGCTGGGGGCCGAGCAGCAGAACCCCTACGCCCTCGCGGAGTCCATTTGGCGCACCAGCAACCGCAATATTTTAGACCTGGGTCGCACGGTTGGTGCGGATCGATATCTCCAGGTGATTTACGAAGATCTCGTCCGTGACCCCCGCAAAGTTTTGACAAATATTTGTGATTTCCTGGGGGTGGACTTTGACGAAGCGCTCCTCAATCCCTACAGCGGCGATCGCCTTACCGATGGCCTCCACCAACAGTCCATGGGCGTCGGGGATCCCAATTTCCTCCAGCACAAAACCATTGATCCGGCCCTCGCCGACAAATGGCGCTCAATTACCCTGCCCGCTGCTCTCCAGCTGGATACGATCCAGTTGGCCGAAACGTTTGCTTACGATCTCCCCCAGGAACCCCAGCTAACACCCCAGACCCAATCCTTGCCCTCGATGGTGGAGCGGTTCGTGACAGTGCGCGGTTTAGAAACCTGTCTCTGTGAGTGGGGCGATCGCCACCAACCATTGGTGCTACTTCTCCACGGCATCCTCGAACAGGGGGCCTCCTGGCAACTCATCGCGCCCCAGTTGGCGGCCCAGGGCTATTGGGTTGTGGCCCCAGACCTGCGTGGTCACGGCAAATCCGCCCATGCCCAGTCCTACAGCATGCTTGATTTTTTGGCTGACGTAGATGCCCTTGCCAAACAATTAGGCGATCGCCCCTTTACCTTGGTGGGCCACTCCATGGGTTCCATCATCGGTGCCATGTATGCAGGAATTCGCCAAACCCAGGTAGAAAAGTTGATCCTCGTTGAAACCATTGTCCCCAACGACATCGACGACGCTGAAACCGGTAATCACCTGACGACCCATCTCGATTACCTCGCCGCGCCCCCCCAACACCCGATCTTCCCCAGCCTAGAAGTGGCCGCCCGTCGCCTCCGCCAAGCCACGCCCCAACTACCCAAAGACCTCTCGGCGTTCCTCACCCAGCGCAGCACCAAATCCGTCGAAAAAGGGGTGCAGTGGCGTTGGGATGCTTTCCTCCGTACCCGGGCGGGCATTGAATTCAATGGCATTAGCAGACGACGTTACCTGGCCCTGCTCAAAGATATCCAAGCGCCGATCACCCTCATCTATGGCGATCAGAGTGAATTTAACCGCCCTGCTGATCTCCAGGCGATCCAAGCGGCTCTCCCCCAGGCCCAACGTTTAACGGTTGCTGGCGGCCATAACCTCCATTTTGAGAATCCCCAGGCGATCGCCCAAATTGTTTATCAACAACTCCAGACCCCTGTACCCAAAACACAATAA SEQ ID NO: 18 Amino AcidEngineered NonA with SafB Interior Acyl Binding Pocket domain (underlined)MVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLIFTSFHMNDETIIFSWLPPHHDMGLIGCILTPIYGGIQAIMMSPFSFLQNPLSWLKHITKYKATISGSPNFAYDYCVKRIREEKKEGLDLSSWVTAFNGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQSEQ ID NO: 19 NucleotideEngineered NonA with mycA Interior Acyl Binding Pocket domain (underlined)ATGGTTGGTCAATTTGCAAATTTCGTCGATCTGCTCCAGTACAGAGCTAAACTTCAGGCGCGGAAAACCGTGTTTAGTTTTCTGGCTGATGGCGAAGCGGAATCTGCGGCCCTGACCTACGGAGAATTAGACCAAAAAGCCCAGGCGATCGCCGCTTTTTTGCAAGCTAACCAGGCTCAAGGGCAACGGGCATTATTACTTTATCCACCGGGTTTAGAGTTTATCGGTGCCTTTTTGGGATGTTTGTATGCTGGTGTTGTTGCGGTGCCAGCTTACCCACCACGGCCGAATAAATCCTTTGACCGCCTCCATAGCATTATCCAAGATGCCCAGGCAAAATTTGCCCTCACCACAACAGAACTTAAAGATAAAATTGCCGATCGCCTCGAAGCTTTAGAAGGTACGGATTTTCATTGTTTGGCTACAGATCAAGTTGAATTAATTTCAGGAAAAAATTGGCAAAAACCGAACATTTCCGGCACAGATCTCGCTTTTTTGCAATACACCAGTGGCTCCACGGGCGATCCTAAAGGAGTGATGGTTTCCCACCACAATTTGATCCACAACTCCGGCTTGATCCGGAATGCGCTGGCTATCGACTTAAAAGATACTCTTTTATCTTGGATGCCCTTAACCCATGACATGGGGCTCATAGCTTGCCACCTTGTTCCTGCCTTAGCCGGAATCAATCAAAATTTAATGCCGACAGAATTATTTATTCGAAGACCTATTCTCTGGATGAAAAAAGCTCATGAACATAAAGCCAGCATTCTATCCTCACCTAATTTTGGATACAATTACTTTCTTAAATTTCTGAAAGACAATAAAAGTTACGACTGGGATTTATCCCATATCAGGGTCATTGCAAACGGGGCCGAACCGATCCGCGCTGTGACCCTCGAAAATTTTGCGAAAACCTTCGCTACAGCAGGCTTTCAAAAATCAGCATTTTATCCCTGTTATGGTATGGCTGAAACCACCCTGATCGTTTCCGGTGGTAATGGTCGTGCCCAGCTTCCCCAGGAAATTATCGTCAGCAAACAGGGCATCGAAGCAAACCAAGTTCGCCCTGCCCAAGGGACAGAAACAACGGTGACCTTGGTCGGCAGTGGTGAAGTGATTGGCGACCAAATTGTCAAAATTGTTGACCCCCAGGCTTTAACAGAATGTACCGTCGGTGAAATTGGCGAAGTATGGGTTAAGGGCGAAAGTGTTGCCCAGGGCTATTGGCAAAAGCCAGACCTCACCCAGCAACAATTCCAGGGAAACGTCGGTGCAGAAACGGGCTTTTTACGCACGGGCGATCTGGGTTTTTTGCAAGGTGGCGAACTGTATATTACGGGTCGTTTAAAGGATCTCCTGATTATCCGGGGGCGCAACCACTATCCCCAGGACATTGAATTAACCGTCGAAGTGGCCCATCCCGCTTTACGACAGGGGGCCGGAGCCGCTGTATCAGTAGACGTTAACGGGGAAGAACAGTTAGTCATTGTCCAGGAAGTTGAGCGTAAATATGCCCGCAAATTAAATGTCGCGGCAGTAGCCCAAGCTATTCGTGGGGCGATCGCCGCCGAACATCAACTGCAACCCCAGGCCATTTGTTTTATTAAACCCGGTAGCATTCCCAAAACATCCAGCGGGAAGATTCGTCGCCATGCCTGCAAAGCTGGTTTTCTAGACGGAAGCTTGGCTGTGGTTGGGGAGTGGCAACCCAGCCACCAAAAAGAAGGAAAAGGAATTGGGACACAAGCCGTTACCCCTTCTACGACAACATCAACGAATTTTCCCCTGCCTGACCAGCACCAACAGCAAATTGAAGCCTGGCTTAAGGATAATATTGCCCATCGCCTCGGCATTACGCCCCAACAATTAGACGAAACGGAACCCTTTGCAAGTTATGGGCTGGATTCAGTGCAAGCAGTACAGGTCACAGCCGACTTAGAGGATTGGCTAGGTCGAAAATTAGACCCCACTCTGGCCTACGATTATCCGACCATTCGCACCCTGGCTCAGTTTTTGGTCCAGGGTAATCAAGCGCTAGAGAAAATACCACAGGTGCCGAAAATTCAGGGCAAAGAAATTGCCGTGGTGGGTCTCAGTTGTCGTTTTCCCCAAGCTGACAACCCCGAAGCTTTTTGGGAATTATTACGTAATGGTAAAGATGGAGTTCGCCCCCTTAAAACTCGCTGGGCCACGGGAGAATGGGGTGGTTTTTTAGAAGATATTGACCAGTTTGAGCCGCAATTTTTTGGCATTTCCCCCCGGGAAGCGGAACAAATGGATCCCCAGCAACGCTTACTGTTAGAAGTAACCTGGGAAGCCTTGGAACGGGCAAATATTCCGGCAGAAAGTTTACGCCATTCCCAAACGGGGGTTTTTGTCGGCATTAGTAATAGTGATTATGCCCAGTTGCAGGTGCGGGAAAACAATCCGATCAATCCCTACATGGGGACGGGCAACGCCCACAGTATTGCTGCGAATCGTCTGTCTTATTTCCTCGATCTCCGGGGCGTTTCTCTGAGCATCGATACGGCCTGTTCCTCTTCTCTGGTGGCGGTACATCTGGCCTGTCAAAGTTTAATCAACGGCGAATCGGAGTTGGCGATCGCCGCCGGGGTGAATTTGATTTTGACCCCCGATGTGACCCAGACTTTTACCCAGGCGGGCATGATGAGTAAGACGGGCCGTTGCCAGACCTTTGATGCCGAGGCTGATGGCTATGTGCGGGGCGAAGGTTGTGGGGTCGTTCTCCTCAAACCCCTGGCCCAGGCAGAACGGGACGGGGATAATATTCTCGCGGTGATCCACGGTTCGGCGGTGAATCAAGATGGACGCAGTAACGGTTTGACGGCTCCCAACGGGCGATCGCAACAGGCCGTTATTCGCCAAGCCCTGGCCCAAGCCGGCATTACCGCCGCCGATTTAGCTTACCTAGAGGCCCACGGCACCGGCACGCCCCTGGGTGATCCCATTGAAATTAATTCCCTGAAGGCGGTTTTACAAACGGCGCAGCGGGAACAGCCCTGTGTGGTGGGTTCTGTGAAAACAAACATTGGTCACCTCGAGGCAGCGGCGGGCATCGCGGGCTTAATCAAGGTGATTTTGTCCCTAGAGCATGGAATGATTCCCCAACATTTGCATTTTAAGCAGCTCAATCCCCGCATTGATCTAGACGGTTTAGTGACCATTGCGAGCAAAGATCAGCCTTGGTCAGGCGGGTCACAAAAACGGTTTGCTGGGGTAAGTTCCTTTGGGTTTGGTGGCACCAATGCCCACGTGATTGTCGGGGACTATGCTCAACAAAAATCTCCCCTTGCTCCTCCGGCTACCCAAGACCGCCCTTGGCATTTGCTGACCCTTTCTGCTAAAAATGCCCAGGCCTTAAATGCCCTGCAAAAAAGCTATGGAGACTATCTGGCCCAACATCCCAGCGTTGACCCACGCGATCTCTGTTTGTCTGCCAATACCGGGCGATCGCCCCTCAAAGAACGTCGTTTTTTTGTCTTTAAACAAGTCGCCGATTTACAACAAACTCTCAATCAAGATTTTCTGGCCCAACCACGCCTCAGTTCCCCCGCAAAAATTGCCTTTTTGTTTACGGGGCAAGGTTCCCAATACTACGGCATGGGGCAACAACTGTACCAAACCAGCCCAGTATTTCGGCAAGTGCTGGATGAGTGCGATCGCCTCTGGCAGACCTATTCCCCCGAAGCCCCTGCCCTCACCGACCTGCTGTACGGTAACCATAACCCTGACCTCGTCCACGAAACTGTCTATACCCAGCCCCTCCTCTTTGCTGTTGAATATGCGATCGCCCAACTATGGTTAAGCTGGGGCGTGACGCCAGACTTTTGCATGGGCCATAGCGTCGGCGAATATGTCGCGGCTTGTCTGGCGGGGGTATTTTCCCTGGCAGACGGCATGAAATTAATTACGGCCCGGGGCAAACTGATGCACGCCCTACCCAGCAATGGCAGTATGGCGGCGGTCTTTGCCGATAAAACGGTCATCAAACCCTACCTATCGGAGCATTTGACCGTCGGAGCCGAAAACGGTTCCCATTTGGTGCTATCAGGAAAGACCCCCTGCCTCGAAGCCAGTATTCACAAACTCCAAAGCCAAGGGATCAAAACCAAACCCCTCAAGGTTTCCCATGCTTTCCACTCCCCTTTGATGGCTCCCATGCTGGCAGAGTTTCGGGAAATTGCTGAACAAATTACTTTCCACCCGCCGCGTATCCCGCTCATTTCCAATGTCACGGGCGGCCAGATTGAAGCGGAAATTGCCCAGGCCGACTATTGGGTTAAGCACGTTTCGCAACCCGTCAAATTTGTCCAGAGCATCCAAACCCTGGCCCAAGCGGGTGTCAATGTTTATCTCGAAATCGGCGTAAAACCAGTGCTCCTGAGTATGGGACGCCATTGCTTAGCTGAACAAGAAGCGGTTTGGTTGCCCAGTTTACGTCCCCATAGTGAGCCTTGGCCGGAAATTTTGACCAGTCTCGGCAAACTGTATGAGCAAGGGCTAAACATTGACTGGCAGACCGTGGAAGCTGGCGATCGCCGCCGGAAACTGATTCTGCCCACCTATCCCTTCCAACGGCAACGATATTGGTTTAATCAAGGCTCTTGGCAAACTGTTGAGACCGAATCTGTGAACCCAGGCCCTGACGATCTCAATGATTGGTTGTATCAGGTGGCGTGGACGCCCCTGGACACTTTGCCCCCGGCCCCTGAACCGTCGGCTAAGCTGTGGTTAATCTTGGGCGATCGCCATGATCACCAGCCCATTGAAGCCCAATTTAAAAACGCCCAGCGGGTGTATCTCGGCCAAAGCAATCATTTTCCGACGAATGCCCCCTGGGAAGTATCTGCCGATGCGTTGGATAATTTATTTACTCACGTCGGCTCCCAAAATTTAGCAGGCATCCTTTACCTGTGTCCCCCAGGGGAAGACCCAGAAGACCTAGATGAAATTCAAAAGCAAACCAGTGGCTTCGCCCTCCAACTGATCCAAACCCTGTATCAACAAAAGATCGCGGTTCCCTGCTGGTTTGTGACCCACCAGAGCCAACGGGTGCTTGAAACCGATGCTGTCACCGGATTTGCCCAAGGGGGATTATGGGGACTCGCCCAGGCGATCGCCCTCGAACATCCAGAGTTGTGGGGGGGAATTATTGATGTCGATGACAGCCTGCCAAATTTTGCCCAGATTTGCCAACAAAGACAGGTGCAGCAGTTGGCCGTGCGGCACCAAAAACTCTACGGGGCACAGCTCAAAAAGCAACCGTCACTGCCCCAGAAAAATCTCCAGATTCAACCCCAACAGACCTATCTAGTGACAGGGGGACTGGGGGCCATTGGCCGTAAAATTGCCCAATGGCTAGCCGCAGCAGGAGCAGAAAAAGTAATTCTCGTCAGCCGGCGCGCTCCGGCAGCGGATCAGCAGACGTTACCGACCAATGCGGTGGTTTATCCTTGCGATTTAGCCGACGCAGCCCAGGTGGCAAAGCTGTTTCAAACCTATCCCCACATCAAAGGAATTTTCCATGCGGCGGGTACCTTAGCTGATGGTTTGCTGCAACAACAAACTTGGCAAAAGTTCCAGACCGTCGCCGCCGCCAAAATGAAAGGGACATGGCATCTGCACCGCCATAGTCAAAAGCTCGATCTGGATTTTTTTGTGTTGTTTTCCTCTGTGGCAGGGGTGCTCGGTTCACCGGGACAGGGGAATTATGCCGCCGCAAACCGGGGCATGGCGGCGATCGCCCAATATCGACAAGCCCAAGGTTTACCCGCCCTGGCGATCCATTGGGGGCCTTGGGCCGAAGGGGGAATGGCCAACTCCCTCAGCAACCAAAATTTAGCGTGGCTGCCGCCCCCCCAGGGACTAACAATCCTCGAAAAAGTCTTGGGCGCCCAGGGGGAAATGGGGGTCTTTAAACCGGACTGGCAAAACCTGGCCAAACAGTTCCCCGAATTTGCCAAAACCCATTACTTTGCAGCCGTTATTCCCTCTGCTGAGGCTGTGCCCCCAACGGCTTCAATTTTTGACAAATTAATCAACCTAGAAGCTTCTCAGCGGGCTGACTATCTACTGGATTATCTGCGGCGGTCTGTGGCGCAAATCCTCAAGTTAGAAATTGAGCAAATTCAAAGCCACGATAGCCTGTTGGATCTGGGCATGGATTCGTTGATGATCATGGAGGCGATCGCCAGCCTCAAGCAGGATTTACAACTGATGTTGTACCCCAGGGAAATCTACGAACGGCCCAGACTTGATGTGTTGACGGCCTATCTAGCGGCGGAATTCACCAAGGCCCATGATTCTGAAGCAGCAACGGCGGCAGCAGCGATTCCCTCCCAAAGCCTTTCGGTCAAAACAAAAAAACAGTGGCAAAAACCTGACCACAAAAACCCGAATCCCATTGCCTTTATCCTCTCTAGCCCCCGGTCGGGTTCGACGTTGCTGCGGGTGATGTTAGCCGGACATCCGGGGTTATATTCGCCGCCAGAGCTGCATTTGCTCCCCTTTGAGACTATGGGCGATCGCCACCAGGAATTGGGTCTATCCCACCTCGGCGAAGGGTTACAACGGGCCTTAATGGATCTAGAAAACCTCACCCCAGAGGCAAGCCAGGCGAAGGTCAACCAATGGGTCAAAGCGAATACACCCATTGCAGACATCTATGCCTATCTCCAACGGCAGGCGGAACAACGTTTACTCATCGACAAATCTCCCAGCTACGGCAGCGATCGCCATATTCTAGACCACAGCGAAATCCTCTTTGACCAGGCCAAATATATCCATCTGGTACGCCATCCCTACGCGGTGATTGAATCCTTTACCCGACTGCGGATGGATAAACTGCTGGGGGCCGAGCAGCAGAACCCCTACGCCCTCGCGGAGTCCATTTGGCGCACCAGCAACCGCAATATTTTAGACCTGGGTCGCACGGTTGGTGCGGATCGATATCTCCAGGTGATTTACGAAGATCTCGTCCGTGACCCCCGCAAAGTTTTGACAAATATTTGTGATTTCCTGGGGGTGGACTTTGACGAAGCGCTCCTCAATCCCTACAGCGGCGATCGCCTTACCGATGGCCTCCACCAACAGTCCATGGGCGTCGGGGATCCCAATTTCCTCCAGCACAAAACCATTGATCCGGCCCTCGCCGACAAATGGCGCTCAATTACCCTGCCCGCTGCTCTCCAGCTGGATACGATCCAGTTGGCCGAAACGTTTGCTTACGATCTCCCCCAGGAACCCCAGCTAACACCCCAGACCCAATCCTTGCCCTCGATGGTGGAGCGGTTCGTGACAGTGCGCGGTTTAGAAACCTGTCTCTGTGAGTGGGGCGATCGCCACCAACCATTGGTGCTACTTCTCCACGGCATCCTCGAACAGGGGGCCTCCTGGCAACTCATCGCGCCCCAGTTGGCGGCCCAGGGCTATTGGGTTGTGGCCCCAGACCTGCGTGGTCACGGCAAATCCGCCCATGCCCAGTCCTACAGCATGCTTGATTTTTTGGCTGACGTAGATGCCCTTGCCAAACAATTAGGCGATCGCCCCTTTACCTTGGTGGGCCACTCCATGGGTTCCATCATCGGTGCCATGTATGCAGGAATTCGCCAAACCCAGGTAGAAAAGTTGATCCTCGTTGAAACCATTGTCCCCAACGACATCGACGACGCTGAAACCGGTAATCACCTGACGACCCATCTCGATTACCTCGCCGCGCCCCCCCAACACCCGATCTTCCCCAGCCTAGAAGTGGCCGCCCGTCGCCTCCGCCAAGCCACGCCCCAACTACCCAAAGACCTCTCGGCGTTCCTCACCCAGCGCAGCACCAAATCCGTCGAAAAAGGGGTGCAGTGGCGTTGGGATGCTTTCCTCCGTACCCGGGCGGGCATTGAATTCAATGGCATTAGCAGACGACGTTACCTGGCCCTGCTCAAAGATATCCAAGCGCCGATCACCCTCATCTATGGCGATCAGAGTGAATTTAACCGCCCTGCTGATCTCCAGGCGATCCAAGCGGCTCTCCCCCAGGCCCAACGTTTAACGGTTGCTGGCGGCCATAACCTCCATTTTGAGAATCCCCAGGCGATCGCCCAAATTGTTTATCAACAACTCCAGACCCCTGTACCCAAAACACAATAA SEQ ID NO: 20 Amino AcidEngineered NonA with MycA Interior Acyl Binding Pocket domain (underlined)MVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLIRNALAIDLKDTLLSWMPLTHDMGLIACHLVPALAGINQNLMPTELFIRRPILWMKKAHEHKASILSSPNFGYNYFLKFLKDNKSYDWDLSHIRVIANGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQSEQ ID NO: 21 NucleotideEngineered NonA with dptE Interior Acyl Binding Pocket domain (underlined)ATGGTTGGTCAATTTGCAAATTTCGTCGATCTGCTCCAGTACAGAGCTAAACTTCAGGCGCGGAAAACCGTGTTTAGTTTTCTGGCTGATGGCGAAGCGGAATCTGCGGCCCTGACCTACGGAGAATTAGACCAAAAAGCCCAGGCGATCGCCGCTTTTTTGCAAGCTAACCAGGCTCAAGGGCAACGGGCATTATTACTTTATCCACCGGGTTTAGAGTTTATCGGTGCCTTTTTGGGATGTTTGTATGCTGGTGTTGTTGCGGTGCCAGCTTACCCACCACGGCCGAATAAATCCTTTGACCGCCTCCATAGCATTATCCAAGATGCCCAGGCAAAATTTGCCCTCACCACAACAGAACTTAAAGATAAAATTGCCGATCGCCTCGAAGCTTTAGAAGGTACGGATTTTCATTGTTTGGCTACAGATCAAGTTGAATTAATTTCAGGAAAAAATTGGCAAAAACCGAACATTTCCGGCACAGATCTCGCTTTTTTGCAATACACCAGTGGCTCCACGGGCGATCCTAAAGGAGTGATGGTTTCCCACCACAATTTGATCCACAACTCCGGCTTGCTCGCCGAGGCCTGCGAGCTGACCGCCGCCACTCCCATGGGCGGCTGGCTGCCCATGTACCACGACATGGGGCTCCTGGGCACGCTGACACCGGCCCTGTACCTCGGCACCACGTGCGTGCTGATGAGCTCCACGGCATTCATCAAACGGCCGCACCTGTGGCTACGGACCATCGACCGGTTCGGCCTGGTCTGGTCGTCGGCTCCCGACTTCGCGTACGACATGTGTCTGAAGCGCGTCACCGACGAGCAGATCGCCGGGCTGGACCTGTCCCGCTGGCGGTGGGCCGGCAACGGGGCCGAACCGATCCGCGCTGTGACCCTCGAAAATTTTGCGAAAACCTTCGCTACAGCAGGCTTTCAAAAATCAGCATTTTATCCCTGTTATGGTATGGCTGAAACCACCCTGATCGTTTCCGGTGGTAATGGTCGTGCCCAGCTTCCCCAGGAAATTATCGTCAGCAAACAGGGCATCGAAGCAAACCAAGTTCGCCCTGCCCAAGGGACAGAAACAACGGTGACCTTGGTCGGCAGTGGTGAAGTGATTGGCGACCAAATTGTCAAAATTGTTGACCCCCAGGCTTTAACAGAATGTACCGTCGGTGAAATTGGCGAAGTATGGGTTAAGGGCGAAAGTGTTGCCCAGGGCTATTGGCAAAAGCCAGACCTCACCCAGCAACAATTCCAGGGAAACGTCGGTGCAGAAACGGGCTTTTTACGCACGGGCGATCTGGGTTTTTTGCAAGGTGGCGAACTGTATATTACGGGTCGTTTAAAGGATCTCCTGATTATCCGGGGGCGCAACCACTATCCCCAGGACATTGAATTAACCGTCGAAGTGGCCCATCCCGCTTTACGACAGGGGGCCGGAGCCGCTGTATCAGTAGACGTTAACGGGGAAGAACAGTTAGTCATTGTCCAGGAAGTTGAGCGTAAATATGCCCGCAAATTAAATGTCGCGGCAGTAGCCCAAGCTATTCGTGGGGCGATCGCCGCCGAACATCAACTGCAACCCCAGGCCATTTGTTTTATTAAACCCGGTAGCATTCCCAAAACATCCAGCGGGAAGATTCGTCGCCATGCCTGCAAAGCTGGTTTTCTAGACGGAAGCTTGGCTGTGGTTGGGGAGTGGCAACCCAGCCACCAAAAAGAAGGAAAAGGAATTGGGACACAAGCCGTTACCCCTTCTACGACAACATCAACGAATTTTCCCCTGCCTGACCAGCACCAACAGCAAATTGAAGCCTGGCTTAAGGATAATATTGCCCATCGCCTCGGCATTACGCCCCAACAATTAGACGAAACGGAACCCTTTGCAAGTTATGGGCTGGATTCAGTGCAAGCAGTACAGGTCACAGCCGACTTAGAGGATTGGCTAGGTCGAAAATTAGACCCCACTCTGGCCTACGATTATCCGACCATTCGCACCCTGGCTCAGTTTTTGGTCCAGGGTAATCAAGCGCTAGAGAAAATACCACAGGTGCCGAAAATTCAGGGCAAAGAAATTGCCGTGGTGGGTCTCAGTTGTCGTTTTCCCCAAGCTGACAACCCCGAAGCTTTTTGGGAATTATTACGTAATGGTAAAGATGGAGTTCGCCCCCTTAAAACTCGCTGGGCCACGGGAGAATGGGGTGGTTTTTTAGAAGATATTGACCAGTTTGAGCCGCAATTTTTTGGCATTTCCCCCCGGGAAGCGGAACAAATGGATCCCCAGCAACGCTTACTGTTAGAAGTAACCTGGGAAGCCTTGGAACGGGCAAATATTCCGGCAGAAAGTTTACGCCATTCCCAAACGGGGGTTTTTGTCGGCATTAGTAATAGTGATTATGCCCAGTTGCAGGTGCGGGAAAACAATCCGATCAATCCCTACATGGGGACGGGCAACGCCCACAGTATTGCTGCGAATCGTCTGTCTTATTTCCTCGATCTCCGGGGCGTTTCTCTGAGCATCGATACGGCCTGTTCCTCTTCTCTGGTGGCGGTACATCTGGCCTGTCAAAGTTTAATCAACGGCGAATCGGAGTTGGCGATCGCCGCCGGGGTGAATTTGATTTTGACCCCCGATGTGACCCAGACTTTTACCCAGGCGGGCATGATGAGTAAGACGGGCCGTTGCCAGACCTTTGATGCCGAGGCTGATGGCTATGTGCGGGGCGAAGGTTGTGGGGTCGTTCTCCTCAAACCCCTGGCCCAGGCAGAACGGGACGGGGATAATATTCTCGCGGTGATCCACGGTTCGGCGGTGAATCAAGATGGACGCAGTAACGGTTTGACGGCTCCCAACGGGCGATCGCAACAGGCCGTTATTCGCCAAGCCCTGGCCCAAGCCGGCATTACCGCCGCCGATTTAGCTTACCTAGAGGCCCACGGCACCGGCACGCCCCTGGGTGATCCCATTGAAATTAATTCCCTGAAGGCGGTTTTACAAACGGCGCAGCGGGAACAGCCCTGTGTGGTGGGTTCTGTGAAAACAAACATTGGTCACCTCGAGGCAGCGGCGGGCATCGCGGGCTTAATCAAGGTGATTTTGTCCCTAGAGCATGGAATGATTCCCCAACATTTGCATTTTAAGCAGCTCAATCCCCGCATTGATCTAGACGGTTTAGTGACCATTGCGAGCAAAGATCAGCCTTGGTCAGGCGGGTCACAAAAACGGTTTGCTGGGGTAAGTTCCTTTGGGTTTGGTGGCACCAATGCCCACGTGATTGTCGGGGACTATGCTCAACAAAAATCTCCCCTTGCTCCTCCGGCTACCCAAGACCGCCCTTGGCATTTGCTGACCCTTTCTGCTAAAAATGCCCAGGCCTTAAATGCCCTGCAAAAAAGCTATGGAGACTATCTGGCCCAACATCCCAGCGTTGACCCACGCGATCTCTGTTTGTCTGCCAATACCGGGCGATCGCCCCTCAAAGAACGTCGTTTTTTTGTCTTTAAACAAGTCGCCGATTTACAACAAACTCTCAATCAAGATTTTCTGGCCCAACCACGCCTCAGTTCCCCCGCAAAAATTGCCTTTTTGTTTACGGGGCAAGGTTCCCAATACTACGGCATGGGGCAACAACTGTACCAAACCAGCCCAGTATTTCGGCAAGTGCTGGATGAGTGCGATCGCCTCTGGCAGACCTATTCCCCCGAAGCCCCTGCCCTCACCGACCTGCTGTACGGTAACCATAACCCTGACCTCGTCCACGAAACTGTCTATACCCAGCCCCTCCTCTTTGCTGTTGAATATGCGATCGCCCAACTATGGTTAAGCTGGGGCGTGACGCCAGACTTTTGCATGGGCCATAGCGTCGGCGAATATGTCGCGGCTTGTCTGGCGGGGGTATTTTCCCTGGCAGACGGCATGAAATTAATTACGGCCCGGGGCAAACTGATGCACGCCCTACCCAGCAATGGCAGTATGGCGGCGGTCTTTGCCGATAAAACGGTCATCAAACCCTACCTATCGGAGCATTTGACCGTCGGAGCCGAAAACGGTTCCCATTTGGTGCTATCAGGAAAGACCCCCTGCCTCGAAGCCAGTATTCACAAACTCCAAAGCCAAGGGATCAAAACCAAACCCCTCAAGGTTTCCCATGCTTTCCACTCCCCTTTGATGGCTCCCATGCTGGCAGAGTTTCGGGAAATTGCTGAACAAATTACTTTCCACCCGCCGCGTATCCCGCTCATTTCCAATGTCACGGGCGGCCAGATTGAAGCGGAAATTGCCCAGGCCGACTATTGGGTTAAGCACGTTTCGCAACCCGTCAAATTTGTCCAGAGCATCCAAACCCTGGCCCAAGCGGGTGTCAATGTTTATCTCGAAATCGGCGTAAAACCAGTGCTCCTGAGTATGGGACGCCATTGCTTAGCTGAACAAGAAGCGGTTTGGTTGCCCAGTTTACGTCCCCATAGTGAGCCTTGGCCGGAAATTTTGACCAGTCTCGGCAAACTGTATGAGCAAGGGCTAAACATTGACTGGCAGACCGTGGAAGCTGGCGATCGCCGCCGGAAACTGATTCTGCCCACCTATCCCTTCCAACGGCAACGATATTGGTTTAATCAAGGCTCTTGGCAAACTGTTGAGACCGAATCTGTGAACCCAGGCCCTGACGATCTCAATGATTGGTTGTATCAGGTGGCGTGGACGCCCCTGGACACTTTGCCCCCGGCCCCTGAACCGTCGGCTAAGCTGTGGTTAATCTTGGGCGATCGCCATGATCACCAGCCCATTGAAGCCCAATTTAAAAACGCCCAGCGGGTGTATCTCGGCCAAAGCAATCATTTTCCGACGAATGCCCCCTGGGAAGTATCTGCCGATGCGTTGGATAATTTATTTACTCACGTCGGCTCCCAAAATTTAGCAGGCATCCTTTACCTGTGTCCCCCAGGGGAAGACCCAGAAGACCTAGATGAAATTCAAAAGCAAACCAGTGGCTTCGCCCTCCAACTGATCCAAACCCTGTATCAACAAAAGATCGCGGTTCCCTGCTGGTTTGTGACCCACCAGAGCCAACGGGTGCTTGAAACCGATGCTGTCACCGGATTTGCCCAAGGGGGATTATGGGGACTCGCCCAGGCGATCGCCCTCGAACATCCAGAGTTGTGGGGGGGAATTATTGATGTCGATGACAGCCTGCCAAATTTTGCCCAGATTTGCCAACAAAGACAGGTGCAGCAGTTGGCCGTGCGGCACCAAAAACTCTACGGGGCACAGCTCAAAAAGCAACCGTCACTGCCCCAGAAAAATCTCCAGATTCAACCCCAACAGACCTATCTAGTGACAGGGGGACTGGGGGCCATTGGCCGTAAAATTGCCCAATGGCTAGCCGCAGCAGGAGCAGAAAAAGTAATTCTCGTCAGCCGGCGCGCTCCGGCAGCGGATCAGCAGACGTTACCGACCAATGCGGTGGTTTATCCTTGCGATTTAGCCGACGCAGCCCAGGTGGCAAAGCTGTTTCAAACCTATCCCCACATCAAAGGAATTTTCCATGCGGCGGGTACCTTAGCTGATGGTTTGCTGCAACAACAAACTTGGCAAAAGTTCCAGACCGTCGCCGCCGCCAAAATGAAAGGGACATGGCATCTGCACCGCCATAGTCAAAAGCTCGATCTGGATTTTTTTGTGTTGTTTTCCTCTGTGGCAGGGGTGCTCGGTTCACCGGGACAGGGGAATTATGCCGCCGCAAACCGGGGCATGGCGGCGATCGCCCAATATCGACAAGCCCAAGGTTTACCCGCCCTGGCGATCCATTGGGGGCCTTGGGCCGAAGGGGGAATGGCCAACTCCCTCAGCAACCAAAATTTAGCGTGGCTGCCGCCCCCCCAGGGACTAACAATCCTCGAAAAAGTCTTGGGCGCCCAGGGGGAAATGGGGGTCTTTAAACCGGACTGGCAAAACCTGGCCAAACAGTTCCCCGAATTTGCCAAAACCCATTACTTTGCAGCCGTTATTCCCTCTGCTGAGGCTGTGCCCCCAACGGCTTCAATTTTTGACAAATTAATCAACCTAGAAGCTTCTCAGCGGGCTGACTATCTACTGGATTATCTGCGGCGGTCTGTGGCGCAAATCCTCAAGTTAGAAATTGAGCAAATTCAAAGCCACGATAGCCTGTTGGATCTGGGCATGGATTCGTTGATGATCATGGAGGCGATCGCCAGCCTCAAGCAGGATTTACAACTGATGTTGTACCCCAGGGAAATCTACGAACGGCCCAGACTTGATGTGTTGACGGCCTATCTAGCGGCGGAATTCACCAAGGCCCATGATTCTGAAGCAGCAACGGCGGCAGCAGCGATTCCCTCCCAAAGCCTTTCGGTCAAAACAAAAAAACAGTGGCAAAAACCTGACCACAAAAACCCGAATCCCATTGCCTTTATCCTCTCTAGCCCCCGGTCGGGTTCGACGTTGCTGCGGGTGATGTTAGCCGGACATCCGGGGTTATATTCGCCGCCAGAGCTGCATTTGCTCCCCTTTGAGACTATGGGCGATCGCCACCAGGAATTGGGTCTATCCCACCTCGGCGAAGGGTTACAACGGGCCTTAATGGATCTAGAAAACCTCACCCCAGAGGCAAGCCAGGCGAAGGTCAACCAATGGGTCAAAGCGAATACACCCATTGCAGACATCTATGCCTATCTCCAACGGCAGGCGGAACAACGTTTACTCATCGACAAATCTCCCAGCTACGGCAGCGATCGCCATATTCTAGACCACAGCGAAATCCTCTTTGACCAGGCCAAATATATCCATCTGGTACGCCATCCCTACGCGGTGATTGAATCCTTTACCCGACTGCGGATGGATAAACTGCTGGGGGCCGAGCAGCAGAACCCCTACGCCCTCGCGGAGTCCATTTGGCGCACCAGCAACCGCAATATTTTAGACCTGGGTCGCACGGTTGGTGCGGATCGATATCTCCAGGTGATTTACGAAGATCTCGTCCGTGACCCCCGCAAAGTTTTGACAAATATTTGTGATTTCCTGGGGGTGGACTTTGACGAAGCGCTCCTCAATCCCTACAGCGGCGATCGCCTTACCGATGGCCTCCACCAACAGTCCATGGGCGTCGGGGATCCCAATTTCCTCCAGCACAAAACCATTGATCCGGCCCTCGCCGACAAATGGCGCTCAATTACCCTGCCCGCTGCTCTCCAGCTGGATACGATCCAGTTGGCCGAAACGTTTGCTTACGATCTCCCCCAGGAACCCCAGCTAACACCCCAGACCCAATCCTTGCCCTCGATGGTGGAGCGGTTCGTGACAGTGCGCGGTTTAGAAACCTGTCTCTGTGAGTGGGGCGATCGCCACCAACCATTGGTGCTACTTCTCCACGGCATCCTCGAACAGGGGGCCTCCTGGCAACTCATCGCGCCCCAGTTGGCGGCCCAGGGCTATTGGGTTGTGGCCCCAGACCTGCGTGGTCACGGCAAATCCGCCCATGCCCAGTCCTACAGCATGCTTGATTTTTTGGCTGACGTAGATGCCCTTGCCAAACAATTAGGCGATCGCCCCTTTACCTTGGTGGGCCACTCCATGGGTTCCATCATCGGTGCCATGTATGCAGGAATTCGCCAAACCCAGGTAGAAAAGTTGATCCTCGTTGAAACCATTGTCCCCAACGACATCGACGACGCTGAAACCGGTAATCACCTGACGACCCATCTCGATTACCTCGCCGCGCCCCCCCAACACCCGATCTTCCCCAGCCTAGAAGTGGCCGCCCGTCGCCTCCGCCAAGCCACGCCCCAACTACCCAAAGACCTCTCGGCGTTCCTCACCCAGCGCAGCACCAAATCCGTCGAAAAAGGGGTGCAGTGGCGTTGGGATGCTTTCCTCCGTACCCGGGCGGGCATTGAATTCAATGGCATTAGCAGACGACGTTACCTGGCCCTGCTCAAAGATATCCAAGCGCCGATCACCCTCATCTATGGCGATCAGAGTGAATTTAACCGCCCTGCTGATCTCCAGGCGATCCAAGCGGCTCTCCCCCAGGCCCAACGTTTAACGGTTGCTGGCGGCCATAACCTCCATTTTGAGAATCCCCAGGCGATCGCCCAAATTGTTTATCAACAACTCCAGACCCCTGTACCCAAAACACAATAA SEQ ID NO: 22 Amino AcidEngineered NonA with DptE Interior Acyl Binding Pocket domain (underlined)MVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLLAEACELTAATPMGGWLPMYHDMGLLGTLTPALYLGTTCVLMSSTAFIKRPHLWLRTIDRFGLVWSSAPDFAYDMCLKRVTDEQIAGLDLSRWRWAGNGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQ IVYQQLQTPVPKTQSEQ ID NO: 23 nonA_optV6 Nucleotide SequenceThe flanking DNA base pairs added to generate restriction sites are in lowercase. The Interior Acyl Binding Pocket-encoding sequence is underlined.catATGGCAAGCTGGTCCCACCCGCAATTCGAGAAAGAAGTACATCACCATCACCATCATGGCGCAGTGGGCCAGTTTGCGAACTTTGTAGACCTGTTGCAATACCGTGCCAAGCTGCAAGCACGTAAGACCGTCTTTAGCTTCCTGGCGGACGGCGAAGCGGAGAGCGCCGCTCTGACCTATGGTGAGCTGGATCAAAAGGCGCAGGCAATCGCGGCGTTCCTGCAAGCAAATCAGGCACAAGGCCAACGTGCATTGCTGCTGTATCCGCCAGGTCTGGAGTTCATCGGTGCCTTCCTGGGTTGTCTGTATGCGGGTGTCGTCGCGGTTCCGGCATATCCTCCGCGTCCGAACAAGTCCTTCGACCGTTTGCACTCCATCATTCAGGACGCCCAAGCGAAGTTTGCACTGACGACGACCGAGTTGAAGGATAAGATTGCAGACCGTCTGGAAGCGCTGGAGGGTACGGACTTCCATTGCCTGGCGACCGACCAAGTCGAGCTGATCAGCGGCAAAAACTGGCAAAAGCCGAATATCTCCGGTACGGATCTGGCGTTTCTGCAATACACCAGCGGCAGCACGGGTGATCCAAAAGGCGTGATGGTCAGCCACCATAACCTGATTCACAATAGCGGTCTGATTAACCAGGGTTTCCAAGACACCGAAGCGAGCATGGGTGTGTCCTGGCTGCCGCCGTATCACGACATGGGTCTGATTGGCGGCATCCTGCAACCTATCTACGTTGGCGCAACGCAAATCCTGATGCCACCAGTCGCCTTTCTGCAACGTCCGTTCCGCTGGCTGAAGGCGATCAACGATTACCGTGTCAGCACCAGCGGTGCGCCGAACTTTGCTTACGACCTGTGCGCTTCTCAGATTACCCCGGAACAAATCCGCGAGCTGGATCTGAGCTGTTGGCGTCTGGCATTCAGCGGTGCAGAGCCGATTCGCGCTGTCACGCTGGAAAACTTTGCGAAAACGTTCGCAACCGCGGGTTTCCAGAAATCGGCCTTCTACCCTTGTTACGGTATGGCGGAAACCACCCTGATCGTGAGCGGTGGCAATGGCCGTGCCCAACTGCCACAGGAGATCATCGTTAGCAAGCAGGGCATTGAGGCGAACCAAGTGCGTCCGGCTCAAGGCACGGAAACGACCGTGACCCTGGTGGGTAGCGGTGAGGTCATTGGTGACCAGATCGTTAAGATCGTTGACCCTCAAGCGCTGACCGAGTGCACCGTCGGTGAAATTGGCGAGGTGTGGGTTAAAGGTGAAAGCGTTGCTCAGGGCTACTGGCAGAAGCCGGACTTGACGCAGCAGCAGTTCCAGGGTAACGTGGGTGCCGAAACGGGTTTCCTGCGCACCGGCGATCTGGGTTTCCTGCAAGGCGGCGAGCTGTATATCACCGGCCGTCTGAAGGATCTGCTGATCATTCGTGGCCGTAATCACTATCCTCAGGACATTGAGCTGACCGTGGAAGTTGCTCACCCAGCCCTGCGTCAGGGCGCAGGTGCCGCGGTGAGCGTGGACGTTAATGGTGAAGAACAACTGGTGATCGTTCAAGAGGTTGAGCGTAAGTACGCACGCAAGCTGAATGTGGCAGCAGTCGCTCAGGCCATCCGTGGTGCGATTGCGGCAGAGCACCAGTTGCAGCCGCAGGCGATCTGCTTTATCAAACCGGGCAGCATCCCGAAAACTAGCAGCGGCAAAATCCGTCGTCACGCATGTAAGGCCGGTTTTCTGGACGGAAGCTTGGCGGTTGTTGGTGAGTGGCAACCGAGCCATCAGAAAGAGGGCAAAGGTATTGGTACCCAGGCAGTGACCCCGAGCACCACGACGTCCACCAACTTTCCGCTGCCGGATCAACACCAGCAACAGATCGAGGCGTGGCTGAAGGACAACATCGCGCACCGCCTGGGTATTACGCCGCAGCAGTTGGATGAAACGGAACCGTTCGCTTCTTACGGTCTGGACAGCGTTCAAGCAGTCCAGGTCACCGCAGACCTGGAGGACTGGCTGGGCCGCAAGCTGGACCCGACTCTGGCCTATGATTACCCGACCATTCGCACGCTGGCGCAATTCCTGGTTCAGGGCAACCAGGCCTTGGAGAAAATCCCGCAAGTTCCAAAGATTCAGGGTAAAGAGATTGCGGTGGTGGGCCTGAGCTGCCGCTTTCCGCAGGCGGACAATCCGGAGGCGTTCTGGGAACTGTTGCGCAATGGCAAGGATGGCGTGCGTCCGCTGAAAACCCGTTGGGCCACTGGTGAGTGGGGTGGTTTCCTGGAGGATATCGACCAGTTTGAGCCGCAGTTCTTTGGTATTAGCCCGCGTGAGGCGGAGCAAATGGACCCGCAACAGCGTCTGCTGCTGGAGGTCACCTGGGAGGCACTGGAGCGTGCGAATATCCCTGCCGAATCCCTGCGTCACAGCCAGACCGGCGTCTTTGTGGGCATTAGCAACAGCGATTACGCACAACTGCAAGTGCGTGAGAACAACCCGATCAATCCGTACATGGGTACTGGTAACGCACATAGCATCGCGGCGAATCGTCTGAGCTACTTTCTGGATCTGCGCGGTGTCTCCCTGAGCATTGATACCGCGTGTTCTAGCAGCCTGGTCGCAGTTCATCTGGCGTGCCAAAGCCTGATTAACGGCGAGAGCGAGCTGGCGATTGCTGCGGGTGTTAATCTGATTCTGACCCCGGATGTCACGCAAACCTTTACCCAAGCGGGTATGATGAGCAAGACGGGCCGTTGCCAGACGTTTGATGCGGAGGCGGACGGCTACGTGCGCGGTGAAGGCTGCGGCGTTGTTCTGCTGAAACCGCTGGCTCAGGCGGAGCGTGATGGCGACAATATCCTGGCGGTCATCCACGGTAGCGCGGTTAACCAGGACGGTCGCAGCAATGGTCTGACTGCGCCGAACGGCCGCTCTCAGCAAGCGGTTATCCGTCAGGCCCTGGCGCAGGCGGGCATCACCGCGGCAGACCTGGCGTATTTGGAAGCGCATGGTACGGGCACCCCGCTGGGCGACCCGATTGAAATCAACAGCTTGAAAGCAGTGCTGCAAACCGCCCAGCGCGAGCAACCGTGCGTTGTGGGCAGCGTCAAGACGAACATTGGCCACCTGGAGGCAGCAGCGGGTATTGCAGGTCTGATCAAGGTGATTCTGTCCCTGGAGCACGGCATGATTCCGCAACACCTGCACTTTAAGCAACTGAATCCGCGCATCGACCTGGACGGCCTGGTTACCATCGCGAGCAAAGACCAGCCGTGGTCGGGTGGTAGCCAGAAGCGTTTCGCCGGTGTCAGCAGCTTTGGTTTTGGCGGTACGAATGCTCACGTGATTGTTGGTGATTATGCCCAGCAAAAGTCCCCGCTGGCTCCGCCTGCGACCCAAGACCGTCCTTGGCATCTGCTGACTCTGAGCGCGAAGAACGCACAAGCGTTGAACGCGTTGCAAAAGAGCTATGGTGACTACCTGGCGCAACATCCGAGCGTTGACCCTCGCGATCTGTGCCTGAGCGCTAACACTGGTCGCTCTCCGCTGAAAGAACGCCGCTTCTTCGTGTTCAAGCAGGTTGCCGACTTGCAACAAACCCTGAATCAGGACTTTCTGGCGCAGCCGAGGCTGAGCAGCCCAGCCAAGATTGCGTTCCTGTTCACGGGTCAGGGCAGCCAGTACTACGGTATGGGCCAGCAACTGTATCAGACGTCCCCGGTTTTCCGTCAAGTCCTGGATGAATGCGACCGTCTGTGGCAGACGTACAGCCCGGAGGCACCGGCGCTGACCGATCTGCTGTACGGCAATCATAATCCTGACCTGGTTCATGAAACGGTTTACACGCAACCGCTGCTGTTCGCGGTGGAGTATGCTATCGCGCAGTTGTGGTTGAGCTGGGGCGTTACTCCGGATTTCTGCATGGGTCATAGCGTCGGTGAGTATGTGGCGGCCTGCCTGGCGGGTGTGTTTAGCCTGGCGGATGGCATGAAACTGATTACCGCGCGTGGTAAACTGATGCATGCACTGCCGAGCAATGGCAGCATGGCGGCTGTGTTTGCGGACAAAACCGTTATCAAGCCGTATCTGAGCGAACACCTGACCGTCGGCGCAGAAAATGGCAGCCACCTGGTTCTGAGCGGTAAGACCCCTTGTCTGGAAGCATCCATCCACAAACTGCAAAGCCAGGGCATCAAAACCAAGCCTCTGAAAGTCTCCCATGCGTTCCACTCGCCGCTGATGGCGCCGATGCTGGCGGAATTTCGTGAGATCGCCGAACAGATTACGTTCCATCCGCCACGTATCCCGCTGATTAGCAACGTGACGGGTGGTCAAATCGAGGCCGAGATCGCGCAAGCAGACTATTGGGTTAAACATGTTAGCCAGCCGGTGAAGTTCGTTCAGAGCATTCAGACCCTGGCCCAAGCGGGTGTGAATGTGTACCTGGAAATCGGTGTTAAACCAGTCCTGCTGTCTATGGGTCGCCACTGTCTGGCAGAGCAGGAAGCGGTTTGGCTGCCGAGCCTGCGTCCACATAGCGAGCCTTGGCCGGAAATCTTGACTAGTCTGGGCAAACTGTACGAGCAAGGTCTGAATATCGACTGGCAAACGGTTGAAGCCGGTGATCGCCGTCGTAAGCTGATTTTGCCGACCTACCCGTTCCAGCGTCAGCGTTATTGGTTCAACCAAGGTAGCTGGCAAACCGTCGAAACTGAGAGCGTGAATCCAGGCCCGGACGACCTGAATGACTGGCTGTACCAAGTGGCATGGACTCCGCTGGATACGCTGCCGCCTGCACCGGAACCGTCGGCGAAACTGTGGCTGATTCTGGGTGATCGTCACGATCACCAACCGATTGAGGCCCAGTTCAAAAACGCCCAACGTGTGTACCTGGGCCAAAGCAACCACTTTCCGACGAACGCCCCGTGGGAGGTGAGCGCGGACGCACTGGATAACTTGTTTACCCATGTGGGTAGCCAAAACCTGGCAGGCATTCTGTATCTGTGCCCGCCTGGTGAAGATCCGGAGGATCTGGATGAGATTCAGAAACAAACTTCCGGCTTTGCGTTGCAACTGATTCAGACCCTGTATCAGCAGAAAATCGCAGTGCCGTGTTGGTTTGTTACCCATCAAAGCCAGCGTGTGCTGGAAACGGACGCGGTGACGGGTTTTGCCCAAGGTGGTCTGTGGGGTTTGGCGCAAGCGATTGCACTGGAACATCCGGAACTGTGGGGTGGTATCATTGACGTGGATGATAGCCTGCCGAACTTCGCGCAGATTTGTCAGCAACGTCAGGTTCAGCAACTGGCTGTCCGTCACCAGAAACTGTATGGTGCGCAACTGAAGAAGCAGCCGAGCCTGCCGCAGAAGAATCTGCAGATCCAACCTCAACAGACCTACCTGGTCACGGGCGGTTTGGGTGCAATCGGTCGTAAGATTGCGCAGTGGCTGGCGGCTGCGGGTGCTGAGAAAGTTATCCTGGTTAGCCGTCGTGCACCGGCAGCGGATCAACAAACCTTGCCGACCAACGCCGTGGTGTACCCGTGCGATCTGGCGGATGCGGCGCAGGTTGCGAAACTGTTCCAAACCTATCCGCACATTAAGGGTATCTTTCATGCAGCCGGTACGCTGGCTGACGGTTTGCTGCAACAGCAAACCTGGCAGAAATTCCAGACTGTCGCTGCGGCGAAGATGAAGGGCACCTGGCACCTGCATCGCCACTCTCAGAAGTTGGACTTGGATTTCTTTGTTTTGTTTTCGTCTGTTGCGGGTGTGCTGGGTAGCCCTGGTCAAGGCAATTACGCGGCAGCCAACCGTGGCATGGCCGCCATCGCTCAGTACCGCCAGGCTCAAGGTCTGCCGGCACTGGCGATTCACTGGGGCCCTTGGGCGGAAGGTGGTATGGCAAACAGCTTGAGCAACCAAAATCTGGCATGGTTGCCTCCGCCGCAGGGCTTGACCATTCTGGAAAAAGTTTTGGGTGCCCAAGGCGAAATGGGCGTGTTCAAACCGGACTGGCAGAACTTGGCCAAACAATTCCCGGAGTTCGCGAAAACCCATTACTTTGCGGCGGTCATTCCGAGCGCTGAAGCGGTTCCACCGACCGCATCTATCTTCGACAAGCTGATCAATCTGGAAGCGAGCCAGCGCGCAGATTACCTGCTGGACTATCTGCGTAGATCTGTGGCACAAATTCTGAAACTGGAAATTGAGCAGATTCAGAGCCACGACTCCCTGCTGGATCTGGGTATGGATAGCCTGATGATCATGGAGGCGATTGCGTCCCTGAAACAAGACCTGCAACTGATGCTGTATCCGCGTGAGATTTACGAGCGTCCGCGTCTGGATGTTCTGACTGCTTACTTGGCCGCTGAGTTTACCAAAGCGCATGATTCTGAAGCAGCTACCGCCGCAGCTGCGATCCCTAGCCAGAGCCTGAGCGTCAAAACCAAAAAGCAATGGCAGAAACCGGATCATAAGAACCCGAATCCGATTGCGTTCATCCTGAGCAGCCCGCGTAGCGGTAGCACCCTGCTGCGCGTGATGCTGGCCGGTCACCCGGGTCTGTATTCCCCACCGGAACTGCACCTGCTGCCGTTTGAAACGATGGGTGACCGCCACCAGGAACTGGGTCTGTCTCATCTGGGCGAGGGTCTGCAACGTGCCCTGATGGACTTGGAAAATCTGACGCCGGAAGCATCCCAGGCAAAGGTGAACCAATGGGTGAAGGCGAATACGCCGATTGCAGACATCTACGCATACCTGCAACGTCAAGCCGAGCAACGTCTGCTGATTGACAAAAGCCCGAGCTATGGCAGCGACCGCCACATTCTGGATCACAGCGAGATCCTGTTCGATCAGGCGAAATACATCCACCTGGTTCGCCATCCTTATGCGGTCATTGAGAGCTTTACCCGCCTGCGTATGGACAAGCTGCTGGGTGCAGAGCAACAGAATCCGTATGCGCTGGCGGAAAGCATTTGGCGTACCTCGAATCGCAACATTCTGGACTTGGGTCGTACCGTCGGCGCTGACCGCTACCTGCAAGTCATCTACGAGGATCTGGTGCGTGACCCGCGTAAAGTTCTGACCAACATTTGTGATTTTCTGGGTGTCGATTTCGACGAGGCACTGCTGAATCCGTACTCCGGCGACCGCCTGACCGACGGCCTGCACCAGCAAAGCATGGGTGTGGGTGACCCGAACTTCTTGCAGCACAAGACCATTGATCCGGCGCTAGCGGACAAATGGCGTAGCATTACCCTGCCGGCTGCTCTGCAACTGGATACGATTCAACTGGCCGAAACCTTCGCATACGACCTGCCGCAGGAGCCGCAGTTGACGCCGCAGACCCAATCTTTGCCATCGATGGTCGAACGTTTCGTCACGGTTCGCGGCCTGGAAACCTGTCTGTGCGAGTGGGGTGATCGCCATCAACCTCTGGTCTTGCTGTTGCACGGTATCCTGGAGCAAGGCGCGTCTTGGCAGTTGATCGCGCCTCAACTGGCAGCGCAGGGCTATTGGGTCGTCGCTCCGGATCTGCGCGGTCACGGTAAATCTGCGCACGCGCAGTCTTATAGCATGCTGGATTTTCTGGCCGATGTGGACGCGCTGGCCAAACAGTTGGGCGACCGTCCGTTCACCTTGGTTGGTCACAGCATGGGTTCCATCATTGGCGCAATGTATGCTGGCATTCGTCAAACCCAGGTTGAAAAACTGATTCTGGTCGAAACCATCGTCCCGAATGATATTGATGATGCCGAAACCGGCAATCACCTGACCACCCATCTGGATTACCTGGCAGCCCCTCCGCAGCACCCGATCTTTCCGAGCCTGGAAGTTGCGGCTCGTCGTCTGCGCCAAGCCACCCCGCAGTTGCCGAAAGACCTGTCTGCATTTCTGACGCAACGTTCCACGAAGAGCGTCGAGAAGGGTGTGCAGTGGCGCTGGGATGCCTTCTTGCGCACCCGTGCAGGTATCGAGTTTAACGGTATCAGCCGTCGCCGTTATCTGGCGCTGCTGAAAGATATCCAGGCCCCAATTACTTTGATTTACGGTGATCAGTCTGAGTTCAATCGCCCAGCAGACCTGCAAGCGATCCAGGCGGCACTGCCGCAAGCGCAACGCCTGACGGTTGCTGGCGGTCACAACTTGCACTTTGAGAATCCGCAGGCCATCGCCCAGATTGTCTATCAGCAGTTGCAGACACCGGTTCCGAAAACCCAAGGTTTGCACCATCACCACCATCATAGCGCCTGGAGCCACCCGCAGTTTGAAAAGTAAgaattc SEQ ID NO: 24 NonA_optV6Amino Acid Sequence Interior Acyl Binding Pocket is underlined.MASWSHPQFEKEVHHHHHHGAVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLINQGFQDTEASMGVSWLPPYHDMGLIGGILQPIYVGATQILMPPVAFLQRPFRWLKAINDYRVSTSGAPNFAYDLCASQITPEQIRELDLSCWRLAFSGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQGLHHHHHHSAWSHPQFEKSEQ ID NO: 25 Codon-optimized sfp Nucleotide SequenceThe flanking DNA base pairs added to generate restriction sites are in lower case.tcATGAAAATTTACGGCATTTACATGGACCGTCCTTTGAGCCAAGAAGAAAATGAGCGTTTTATGTCGTTCATCAGCCCGGAAAAACGCGAGAAGTGCCGTCGTTTCTATCATAAGGAGGATGCCCATCGCACGCTGCTGGGTGATGTTCTGGTTCGTTCCGTGATCTCCCGCCAATACCAGCTGGACAAAAGCGATATCCGCTTTTCCACCCAGGAGTACGGCAAACCATGTATCCCGGACCTGCCGGACGCTCACTTCAACATTAGCCACAGCGGTCGTTGGGTGATTTGTGCGTTCGATAGCCAGCCGATTGGTATTGACATTGAAAAGACGAAGCCTATTAGCCTGGAGATCGCCAAGCGCTTCTTCAGCAAAACCGAGTATAGCGATCTGCTGGCGAAAGACAAAGACGAGCAAACCGACTACTTTTACCACCTGTGGAGCATGAAAGAAAGCTTTATCAAGCAAGAAGGTAAGGGTTTGAGCTTGCCGCTGGACAGCTTTAGCGTGCGTCTGCATCAGGATGGTCAGGTCAGCATCGAGCTGCCGGACTCTCACTCTCCGTGCTATATTAAAACCTACGAGGTCGATCCGGGCTATAAAATGGCGGTTTGCGCAGCACACCCGGACTTTCCGGAGGATATCACTATGGTGAGCTATGAAGAGTTGCTGTAAgaattcSEQ ID NO: 26 nonA_dptE Nucleotide SequenceThe Interior Acyl Binding Pocket-encoding sequence is underlined.ATGGCAAGCTGGTCCCACCCGCAATTCGAGAAAGAAGTACATCACCATCACCATCATGGCGCAGTGGGCCAGTTTGCGAACTTTGTAGACCTGTTGCAATACCGTGCCAAGCTGCAAGCACGTAAGACCGTCTTTAGCTTCCTGGCGGACGGCGAAGCGGAGAGCGCCGCTCTGACCTATGGTGAGCTGGATCAAAAGGCGCAGGCAATCGCGGCGTTCCTGCAAGCAAATCAGGCACAAGGCCAACGTGCATTGCTGCTGTATCCGCCAGGTCTGGAGTTCATCGGTGCCTTCCTGGGTTGTCTGTATGCGGGTGTCGTCGCGGTTCCGGCATATCCTCCGCGTCCGAACAAGTCCTTCGACCGTTTGCACTCCATCATTCAGGACGCCCAAGCGAAGTTTGCACTGACGACGACCGAGTTGAAGGATAAGATTGCAGACCGTCTGGAAGCGCTGGAGGGTACGGACTTCCATTGCCTGGCGACCGACCAAGTCGAGCTGATCAGCGGCAAAAACTGGCAAAAGCCGAATATCTCCGGTACGGATCTGGCGTTTCTGCAATACACCAGCGGCAGCACGGGTGATCCAAAAGGCGTGATGGTCAGCCACCATAACCTGATTCACAATAGCGGTCTGTTGGCGGAAGCGTGCGAACTGACCGCTGCGACCCCGATGGGCGGTTGGCTGCCGATGTACCATGATATGGGCTTGCTGGGTACTCTGACGCCAGCGTTGTACCTGGGTACTACCTGTGTCCTGATGTCTAGCACCGCCTTCATCAAACGCCCGCATCTGTGGCTGCGCACCATTGATCGCTTTGGTCTGGTTTGGTCTAGCGCTCCGGATTTCGCGTACGATATGTGCCTGAAACGTGTTACCGATGAGCAGATTGCGGGTCTGGATCTGTCTCGCTGGCGCTGGGCGGGTAACGGTGCAGAGCCGATTCGCGCTGTCACGCTGGAAAACTTTGCGAAAACGTTCGCAACCGCGGGTTTCCAGAAATCGGCCTTCTACCCTTGTTACGGTATGGCGGAAACCACCCTGATCGTGAGCGGTGGCAATGGCCGTGCCCAACTGCCACAGGAGATCATCGTTAGCAAGCAGGGCATTGAGGCGAACCAAGTGCGTCCGGCTCAAGGCACGGAAACGACCGTGACCCTGGTGGGTAGCGGTGAGGTCATTGGTGACCAGATCGTTAAGATCGTTGACCCTCAAGCGCTGACCGAGTGCACCGTCGGTGAAATTGGCGAGGTGTGGGTTAAAGGTGAAAGCGTTGCTCAGGGCTACTGGCAGAAGCCGGACTTGACGCAGCAGCAGTTCCAGGGTAACGTGGGTGCCGAAACGGGTTTCCTGCGCACCGGCGATCTGGGTTTCCTGCAAGGCGGCGAGCTGTATATCACCGGCCGTCTGAAGGATCTGCTGATCATTCGTGGCCGTAATCACTATCCTCAGGACATTGAGCTGACCGTGGAAGTTGCTCACCCAGCCCTGCGTCAGGGCGCAGGTGCCGCGGTGAGCGTGGACGTTAATGGTGAAGAACAACTGGTGATCGTTCAAGAGGTTGAGCGTAAGTACGCACGCAAGCTGAATGTGGCAGCAGTCGCTCAGGCCATCCGTGGTGCGATTGCGGCAGAGCACCAGTTGCAGCCGCAGGCGATCTGCTTTATCAAACCGGGCAGCATCCCGAAAACTAGCAGCGGCAAAATCCGTCGTCACGCATGTAAGGCCGGTTTTCTGGACGGAAGCTTGGCGGTTGTTGGTGAGTGGCAACCGAGCCATCAGAAAGAGGGCAAAGGTATTGGTACCCAGGCAGTGACCCCGAGCACCACGACGTCCACCAACTTTCCGCTGCCGGATCAACACCAGCAACAGATCGAGGCGTGGCTGAAGGACAACATCGCGCACCGCCTGGGTATTACGCCGCAGCAGTTGGATGAAACGGAACCGTTCGCTTCTTACGGTCTGGACAGCGTTCAAGCAGTCCAGGTCACCGCAGACCTGGAGGACTGGCTGGGCCGCAAGCTGGACCCGACTCTGGCCTATGATTACCCGACCATTCGCACGCTGGCGCAATTCCTGGTTCAGGGCAACCAGGCCTTGGAGAAAATCCCGCAAGTTCCAAAGATTCAGGGTAAAGAGATTGCGGTGGTGGGCCTGAGCTGCCGCTTTCCGCAGGCGGACAATCCGGAGGCGTTCTGGGAACTGTTGCGCAATGGCAAGGATGGCGTGCGTCCGCTGAAAACCCGTTGGGCCACTGGTGAGTGGGGTGGTTTCCTGGAGGATATCGACCAGTTTGAGCCGCAGTTCTTTGGTATTAGCCCGCGTGAGGCGGAGCAAATGGACCCGCAACAGCGTCTGCTGCTGGAGGTCACCTGGGAGGCACTGGAGCGTGCGAATATCCCTGCCGAATCCCTGCGTCACAGCCAGACCGGCGTCTTTGTGGGCATTAGCAACAGCGATTACGCACAACTGCAAGTGCGTGAGAACAACCCGATCAATCCGTACATGGGTACTGGTAACGCACATAGCATCGCGGCGAATCGTCTGAGCTACTTTCTGGATCTGCGCGGTGTCTCCCTGAGCATTGATACCGCGTGTTCTAGCAGCCTGGTCGCAGTTCATCTGGCGTGCCAAAGCCTGATTAACGGCGAGAGCGAGCTGGCGATTGCTGCGGGTGTTAATCTGATTCTGACCCCGGATGTCACGCAAACCTTTACCCAAGCGGGTATGATGAGCAAGACGGGCCGTTGCCAGACGTTTGATGCGGAGGCGGACGGCTACGTGCGCGGTGAAGGCTGCGGCGTTGTTCTGCTGAAACCGCTGGCTCAGGCGGAGCGTGATGGCGACAATATCCTGGCGGTCATCCACGGTAGCGCGGTTAACCAGGACGGTCGCAGCAATGGTCTGACTGCGCCGAACGGCCGCTCTCAGCAAGCGGTTATCCGTCAGGCCCTGGCGCAGGCGGGCATCACCGCGGCAGACCTGGCGTATTTGGAAGCGCATGGTACGGGCACCCCGCTGGGCGACCCGATTGAAATCAACAGCTTGAAAGCAGTGCTGCAAACCGCCCAGCGCGAGCAACCGTGCGTTGTGGGCAGCGTCAAGACGAACATTGGCCACCTGGAGGCAGCAGCGGGTATTGCAGGTCTGATCAAGGTGATTCTGTCCCTGGAGCACGGCATGATTCCGCAACACCTGCACTTTAAGCAACTGAATCCGCGCATCGACCTGGACGGCCTGGTTACCATCGCGAGCAAAGACCAGCCGTGGTCGGGTGGTAGCCAGAAGCGTTTCGCCGGTGTCAGCAGCTTTGGTTTTGGCGGTACGAATGCTCACGTGATTGTTGGTGATTATGCCCAGCAAAAGTCCCCGCTGGCTCCGCCTGCGACCCAAGACCGTCCTTGGCATCTGCTGACTCTGAGCGCGAAGAACGCACAAGCGTTGAACGCGTTGCAAAAGAGCTATGGTGACTACCTGGCGCAACATCCGAGCGTTGACCCTCGCGATCTGTGCCTGAGCGCTAACACTGGTCGCTCTCCGCTGAAAGAACGCCGCTTCTTCGTGTTCAAGCAGGTTGCCGACTTGCAACAAACCCTGAATCAGGACTTTCTGGCGCAGCCGAGGCTGAGCAGCCCAGCCAAGATTGCGTTCCTGTTCACGGGTCAGGGCAGCCAGTACTACGGTATGGGCCAGCAACTGTATCAGACGTCCCCGGTTTTCCGTCAAGTCCTGGATGAATGCGACCGTCTGTGGCAGACGTACAGCCCGGAGGCACCGGCGCTGACCGATCTGCTGTACGGCAATCATAATCCTGACCTGGTTCATGAAACGGTTTACACGCAACCGCTGCTGTTCGCGGTGGAGTATGCTATCGCGCAGTTGTGGTTGAGCTGGGGCGTTACTCCGGATTTCTGCATGGGTCATAGCGTCGGTGAGTATGTGGCGGCCTGCCTGGCGGGTGTGTTTAGCCTGGCGGATGGCATGAAACTGATTACCGCGCGTGGTAAACTGATGCATGCACTGCCGAGCAATGGCAGCATGGCGGCTGTGTTTGCGGACAAAACCGTTATCAAGCCGTATCTGAGCGAACACCTGACCGTCGGCGCAGAAAATGGCAGCCACCTGGTTCTGAGCGGTAAGACCCCTTGTCTGGAAGCATCCATCCACAAACTGCAAAGCCAGGGCATCAAAACCAAGCCTCTGAAAGTCTCCCATGCGTTCCACTCGCCGCTGATGGCGCCGATGCTGGCGGAATTTCGTGAGATCGCCGAACAGATTACGTTCCATCCGCCACGTATCCCGCTGATTAGCAACGTGACGGGTGGTCAAATCGAGGCCGAGATCGCGCAAGCAGACTATTGGGTTAAACATGTTAGCCAGCCGGTGAAGTTCGTTCAGAGCATTCAGACCCTGGCCCAAGCGGGTGTGAATGTGTACCTGGAAATCGGTGTTAAACCAGTCCTGCTGTCTATGGGTCGCCACTGTCTGGCAGAGCAGGAAGCGGTTTGGCTGCCGAGCCTGCGTCCACATAGCGAGCCTTGGCCGGAAATCTTGACTAGTCTGGGCAAACTGTACGAGCAAGGTCTGAATATCGACTGGCAAACGGTTGAAGCCGGTGATCGCCGTCGTAAGCTGATTTTGCCGACCTACCCGTTCCAGCGTCAGCGTTATTGGTTCAACCAAGGTAGCTGGCAAACCGTCGAAACTGAGAGCGTGAATCCAGGCCCGGACGACCTGAATGACTGGCTGTACCAAGTGGCATGGACTCCGCTGGATACGCTGCCGCCTGCACCGGAACCGTCGGCGAAACTGTGGCTGATTCTGGGTGATCGTCACGATCACCAACCGATTGAGGCCCAGTTCAAAAACGCCCAACGTGTGTACCTGGGCCAAAGCAACCACTTTCCGACGAACGCCCCGTGGGAGGTGAGCGCGGACGCACTGGATAACTTGTTTACCCATGTGGGTAGCCAAAACCTGGCAGGCATTCTGTATCTGTGCCCGCCTGGTGAAGATCCGGAGGATCTGGATGAGATTCAGAAACAAACTTCCGGCTTTGCGTTGCAACTGATTCAGACCCTGTATCAGCAGAAAATCGCAGTGCCGTGTTGGTTTGTTACCCATCAAAGCCAGCGTGTGCTGGAAACGGACGCGGTGACGGGTTTTGCCCAAGGTGGTCTGTGGGGTTTGGCGCAAGCGATTGCACTGGAACATCCGGAACTGTGGGGTGGTATCATTGACGTGGATGATAGCCTGCCGAACTTCGCGCAGATTTGTCAGCAACGTCAGGTTCAGCAACTGGCTGTCCGTCACCAGAAACTGTATGGTGCGCAACTGAAGAAGCAGCCGAGCCTGCCGCAGAAGAATCTGCAGATCCAACCTCAACAGACCTACCTGGTCACGGGCGGTTTGGGTGCAATCGGTCGTAAGATTGCGCAGTGGCTGGCGGCTGCGGGTGCTGAGAAAGTTATCCTGGTTAGCCGTCGTGCACCGGCAGCGGATCAACAAACCTTGCCGACCAACGCCGTGGTGTACCCGTGCGATCTGGCGGATGCGGCGCAGGTTGCGAAACTGTTCCAAACCTATCCGCACATTAAGGGTATCTTTCATGCAGCCGGTACGCTGGCTGACGGTTTGCTGCAACAGCAAACCTGGCAGAAATTCCAGACTGTCGCTGCGGCGAAGATGAAGGGCACCTGGCACCTGCATCGCCACTCTCAGAAGTTGGACTTGGATTTCTTTGTTTTGTTTTCGTCTGTTGCGGGTGTGCTGGGTAGCCCTGGTCAAGGCAATTACGCGGCAGCCAACCGTGGCATGGCCGCCATCGCTCAGTACCGCCAGGCTCAAGGTCTGCCGGCACTGGCGATTCACTGGGGCCCTTGGGCGGAAGGTGGTATGGCAAACAGCTTGAGCAACCAAAATCTGGCATGGTTGCCTCCGCCGCAGGGCTTGACCATTCTGGAAAAAGTTTTGGGTGCCCAAGGCGAAATGGGCGTGTTCAAACCGGACTGGCAGAACTTGGCCAAACAATTCCCGGAGTTCGCGAAAACCCATTACTTTGCGGCGGTCATTCCGAGCGCTGAAGCGGTTCCACCGACCGCATCTATCTTCGACAAGCTGATCAATCTGGAAGCGAGCCAGCGCGCAGATTACCTGCTGGACTATCTGCGTAGATCTGTGGCACAAATTCTGAAACTGGAAATTGAGCAGATTCAGAGCCACGACTCCCTGCTGGATCTGGGTATGGATAGCCTGATGATCATGGAGGCGATTGCGTCCCTGAAACAAGACCTGCAACTGATGCTGTATCCGCGTGAGATTTACGAGCGTCCGCGTCTGGATGTTCTGACTGCTTACTTGGCCGCTGAGTTTACCAAAGCGCATGATTCTGAAGCAGCTACCGCCGCAGCTGCGATCCCTAGCCAGAGCCTGAGCGTCAAAACCAAAAAGCAATGGCAGAAACCGGATCATAAGAACCCGAATCCGATTGCGTTCATCCTGAGCAGCCCGCGTAGCGGTAGCACCCTGCTGCGCGTGATGCTGGCCGGTCACCCGGGTCTGTATTCCCCACCGGAACTGCACCTGCTGCCGTTTGAAACGATGGGTGACCGCCACCAGGAACTGGGTCTGTCTCATCTGGGCGAGGGTCTGCAACGTGCCCTGATGGACTTGGAAAATCTGACGCCGGAAGCATCCCAGGCAAAGGTGAACCAATGGGTGAAGGCGAATACGCCGATTGCAGACATCTACGCATACCTGCAACGTCAAGCCGAGCAACGTCTGCTGATTGACAAAAGCCCGAGCTATGGCAGCGACCGCCACATTCTGGATCACAGCGAGATCCTGTTCGATCAGGCGAAATACATCCACCTGGTTCGCCATCCTTATGCGGTCATTGAGAGCTTTACCCGCCTGCGTATGGACAAGCTGCTGGGTGCAGAGCAACAGAATCCGTATGCGCTGGCGGAAAGCATTTGGCGTACCTCGAATCGCAACATTCTGGACTTGGGTCGTACCGTCGGCGCTGACCGCTACCTGCAAGTCATCTACGAGGATCTGGTGCGTGACCCGCGTAAAGTTCTGACCAACATTTGTGATTTTCTGGGTGTCGATTTCGACGAGGCACTGCTGAATCCGTACTCCGGCGACCGCCTGACCGACGGCCTGCACCAGCAAAGCATGGGTGTGGGTGACCCGAACTTCTTGCAGCACAAGACCATTGATCCGGCGCTAGCGGACAAATGGCGTAGCATTACCCTGCCGGCTGCTCTGCAACTGGATACGATTCAACTGGCCGAAACCTTCGCATACGACCTGCCGCAGGAGCCGCAGTTGACGCCGCAGACCCAATCTTTGCCATCGATGGTCGAACGTTTCGTCACGGTTCGCGGCCTGGAAACCTGTCTGTGCGAGTGGGGTGATCGCCATCAACCTCTGGTCTTGCTGTTGCACGGTATCCTGGAGCAAGGCGCGTCTTGGCAGTTGATCGCGCCTCAACTGGCAGCGCAGGGCTATTGGGTCGTCGCTCCGGATCTGCGCGGTCACGGTAAATCTGCGCACGCGCAGTCTTATAGCATGCTGGATTTTCTGGCCGATGTGGACGCGCTGGCCAAACAGTTGGGCGACCGTCCGTTCACCTTGGTTGGTCACAGCATGGGTTCCATCATTGGCGCAATGTATGCTGGCATTCGTCAAACCCAGGTTGAAAAACTGATTCTGGTCGAAACCATCGTCCCGAATGATATTGATGATGCCGAAACCGGCAATCACCTGACCACCCATCTGGATTACCTGGCAGCCCCTCCGCAGCACCCGATCTTTCCGAGCCTGGAAGTTGCGGCTCGTCGTCTGCGCCAAGCCACCCCGCAGTTGCCGAAAGACCTGTCTGCATTTCTGACGCAACGTTCCACGAAGAGCGTCGAGAAGGGTGTGCAGTGGCGCTGGGATGCCTTCTTGCGCACCCGTGCAGGTATCGAGTTTAACGGTATCAGCCGTCGCCGTTATCTGGCGCTGCTGAAAGATATCCAGGCCCCAATTACTTTGATTTACGGTGATCAGTCTGAGTTCAATCGCCCAGCAGACCTGCAAGCGATCCAGGCGGCACTGCCGCAAGCGCAACGCCTGACGGTTGCTGGCGGTCACAACTTGCACTTTGAGAATCCGCAGGCCATCGCCCAGATTGTCTATCAGCAGTTGCAGACACCGGTTCCGAAAACCCAAGGTTTGCACCATCACCACCATCATAGCGCCTGGAGCCACCCGCAGTTTGAAAAGTAA SEQ ID NO: 27 nonA_safBNucleotide SequenceThe Interior Acyl Binding Pocket-encoding sequence is underlined.ATGGCAAGCTGGTCCCACCCGCAATTCGAGAAAGAAGTACATCACCATCACCATCATGGCGCAGTGGGCCAGTTTGCGAACTTTGTAGACCTGTTGCAATACCGTGCCAAGCTGCAAGCACGTAAGACCGTCTTTAGCTTCCTGGCGGACGGCGAAGCGGAGAGCGCCGCTCTGACCTATGGTGAGCTGGATCAAAAGGCGCAGGCAATCGCGGCGTTCCTGCAAGCAAATCAGGCACAAGGCCAACGTGCATTGCTGCTGTATCCGCCAGGTCTGGAGTTCATCGGTGCCTTCCTGGGTTGTCTGTATGCGGGTGTCGTCGCGGTTCCGGCATATCCTCCGCGTCCGAACAAGTCCTTCGACCGTTTGCACTCCATCATTCAGGACGCCCAAGCGAAGTTTGCACTGACGACGACCGAGTTGAAGGATAAGATTGCAGACCGTCTGGAAGCGCTGGAGGGTACGGACTTCCATTGCCTGGCGACCGACCAAGTCGAGCTGATCAGCGGCAAAAACTGGCAAAAGCCGAATATCTCCGGTACGGATCTGGCGTTTCTGCAATACACCAGCGGCAGCACGGGTGATCCAAAAGGCGTGATGGTCAGCCACCATAACCTGATTCACAATAGCGGTCTGATTTTCACCTCTTTTCACATGAACGATGAAACTATCATTTTCTCGTGGCTGCCGCCACATCACGATATGGGTTTGATTGGCTGCATTCTGACCCCGATTTACGGTGGTATTCAGGCTATCATGATGAGCCCGTTTAGCTTTTTGCAGAACCCGCTGTCCTGGCTGAAACATATCACTAAGTACAAAGCGACCATTTCTGGTAGCCCGAACTTTGCGTACGACTATTGCGTTAAACGCATTCGCGAAGAAAAGAAAGAGGGTCTGGATCTGTCTAGCTGGGTTACCGCGTTCAATGGTGCAGAGCCGATTCGCGCTGTCACGCTGGAAAACTTTGCGAAAACGTTCGCAACCGCGGGTTTCCAGAAATCGGCCTTCTACCCTTGTTACGGTATGGCGGAAACCACCCTGATCGTGAGCGGTGGCAATGGCCGTGCCCAACTGCCACAGGAGATCATCGTTAGCAAGCAGGGCATTGAGGCGAACCAAGTGCGTCCGGCTCAAGGCACGGAAACGACCGTGACCCTGGTGGGTAGCGGTGAGGTCATTGGTGACCAGATCGTTAAGATCGTTGACCCTCAAGCGCTGACCGAGTGCACCGTCGGTGAAATTGGCGAGGTGTGGGTTAAAGGTGAAAGCGTTGCTCAGGGCTACTGGCAGAAGCCGGACTTGACGCAGCAGCAGTTCCAGGGTAACGTGGGTGCCGAAACGGGTTTCCTGCGCACCGGCGATCTGGGTTTCCTGCAAGGCGGCGAGCTGTATATCACCGGCCGTCTGAAGGATCTGCTGATCATTCGTGGCCGTAATCACTATCCTCAGGACATTGAGCTGACCGTGGAAGTTGCTCACCCAGCCCTGCGTCAGGGCGCAGGTGCCGCGGTGAGCGTGGACGTTAATGGTGAAGAACAACTGGTGATCGTTCAAGAGGTTGAGCGTAAGTACGCACGCAAGCTGAATGTGGCAGCAGTCGCTCAGGCCATCCGTGGTGCGATTGCGGCAGAGCACCAGTTGCAGCCGCAGGCGATCTGCTTTATCAAACCGGGCAGCATCCCGAAAACTAGCAGCGGCAAAATCCGTCGTCACGCATGTAAGGCCGGTTTTCTGGACGGAAGCTTGGCGGTTGTTGGTGAGTGGCAACCGAGCCATCAGAAAGAGGGCAAAGGTATTGGTACCCAGGCAGTGACCCCGAGCACCACGACGTCCACCAACTTTCCGCTGCCGGATCAACACCAGCAACAGATCGAGGCGTGGCTGAAGGACAACATCGCGCACCGCCTGGGTATTACGCCGCAGCAGTTGGATGAAACGGAACCGTTCGCTTCTTACGGTCTGGACAGCGTTCAAGCAGTCCAGGTCACCGCAGACCTGGAGGACTGGCTGGGCCGCAAGCTGGACCCGACTCTGGCCTATGATTACCCGACCATTCGCACGCTGGCGCAATTCCTGGTTCAGGGCAACCAGGCCTTGGAGAAAATCCCGCAAGTTCCAAAGATTCAGGGTAAAGAGATTGCGGTGGTGGGCCTGAGCTGCCGCTTTCCGCAGGCGGACAATCCGGAGGCGTTCTGGGAACTGTTGCGCAATGGCAAGGATGGCGTGCGTCCGCTGAAAACCCGTTGGGCCACTGGTGAGTGGGGTGGTTTCCTGGAGGATATCGACCAGTTTGAGCCGCAGTTCTTTGGTATTAGCCCGCGTGAGGCGGAGCAAATGGACCCGCAACAGCGTCTGCTGCTGGAGGTCACCTGGGAGGCACTGGAGCGTGCGAATATCCCTGCCGAATCCCTGCGTCACAGCCAGACCGGCGTCTTTGTGGGCATTAGCAACAGCGATTACGCACAACTGCAAGTGCGTGAGAACAACCCGATCAATCCGTACATGGGTACTGGTAACGCACATAGCATCGCGGCGAATCGTCTGAGCTACTTTCTGGATCTGCGCGGTGTCTCCCTGAGCATTGATACCGCGTGTTCTAGCAGCCTGGTCGCAGTTCATCTGGCGTGCCAAAGCCTGATTAACGGCGAGAGCGAGCTGGCGATTGCTGCGGGTGTTAATCTGATTCTGACCCCGGATGTCACGCAAACCTTTACCCAAGCGGGTATGATGAGCAAGACGGGCCGTTGCCAGACGTTTGATGCGGAGGCGGACGGCTACGTGCGCGGTGAAGGCTGCGGCGTTGTTCTGCTGAAACCGCTGGCTCAGGCGGAGCGTGATGGCGACAATATCCTGGCGGTCATCCACGGTAGCGCGGTTAACCAGGACGGTCGCAGCAATGGTCTGACTGCGCCGAACGGCCGCTCTCAGCAAGCGGTTATCCGTCAGGCCCTGGCGCAGGCGGGCATCACCGCGGCAGACCTGGCGTATTTGGAAGCGCATGGTACGGGCACCCCGCTGGGCGACCCGATTGAAATCAACAGCTTGAAAGCAGTGCTGCAAACCGCCCAGCGCGAGCAACCGTGCGTTGTGGGCAGCGTCAAGACGAACATTGGCCACCTGGAGGCAGCAGCGGGTATTGCAGGTCTGATCAAGGTGATTCTGTCCCTGGAGCACGGCATGATTCCGCAACACCTGCACTTTAAGCAACTGAATCCGCGCATCGACCTGGACGGCCTGGTTACCATCGCGAGCAAAGACCAGCCGTGGTCGGGTGGTAGCCAGAAGCGTTTCGCCGGTGTCAGCAGCTTTGGTTTTGGCGGTACGAATGCTCACGTGATTGTTGGTGATTATGCCCAGCAAAAGTCCCCGCTGGCTCCGCCTGCGACCCAAGACCGTCCTTGGCATCTGCTGACTCTGAGCGCGAAGAACGCACAAGCGTTGAACGCGTTGCAAAAGAGCTATGGTGACTACCTGGCGCAACATCCGAGCGTTGACCCTCGCGATCTGTGCCTGAGCGCTAACACTGGTCGCTCTCCGCTGAAAGAACGCCGCTTCTTCGTGTTCAAGCAGGTTGCCGACTTGCAACAAACCCTGAATCAGGACTTTCTGGCGCAGCCGAGGCTGAGCAGCCCAGCCAAGATTGCGTTCCTGTTCACGGGTCAGGGCAGCCAGTACTACGGTATGGGCCAGCAACTGTATCAGACGTCCCCGGTTTTCCGTCAAGTCCTGGATGAATGCGACCGTCTGTGGCAGACGTACAGCCCGGAGGCACCGGCGCTGACCGATCTGCTGTACGGCAATCATAATCCTGACCTGGTTCATGAAACGGTTTACACGCAACCGCTGCTGTTCGCGGTGGAGTATGCTATCGCGCAGTTGTGGTTGAGCTGGGGCGTTACTCCGGATTTCTGCATGGGTCATAGCGTCGGTGAGTATGTGGCGGCCTGCCTGGCGGGTGTGTTTAGCCTGGCGGATGGCATGAAACTGATTACCGCGCGTGGTAAACTGATGCATGCACTGCCGAGCAATGGCAGCATGGCGGCTGTGTTTGCGGACAAAACCGTTATCAAGCCGTATCTGAGCGAACACCTGACCGTCGGCGCAGAAAATGGCAGCCACCTGGTTCTGAGCGGTAAGACCCCTTGTCTGGAAGCATCCATCCACAAACTGCAAAGCCAGGGCATCAAAACCAAGCCTCTGAAAGTCTCCCATGCGTTCCACTCGCCGCTGATGGCGCCGATGCTGGCGGAATTTCGTGAGATCGCCGAACAGATTACGTTCCATCCGCCACGTATCCCGCTGATTAGCAACGTGACGGGTGGTCAAATCGAGGCCGAGATCGCGCAAGCAGACTATTGGGTTAAACATGTTAGCCAGCCGGTGAAGTTCGTTCAGAGCATTCAGACCCTGGCCCAAGCGGGTGTGAATGTGTACCTGGAAATCGGTGTTAAACCAGTCCTGCTGTCTATGGGTCGCCACTGTCTGGCAGAGCAGGAAGCGGTTTGGCTGCCGAGCCTGCGTCCACATAGCGAGCCTTGGCCGGAAATCTTGACTAGTCTGGGCAAACTGTACGAGCAAGGTCTGAATATCGACTGGCAAACGGTTGAAGCCGGTGATCGCCGTCGTAAGCTGATTTTGCCGACCTACCCGTTCCAGCGTCAGCGTTATTGGTTCAACCAAGGTAGCTGGCAAACCGTCGAAACTGAGAGCGTGAATCCAGGCCCGGACGACCTGAATGACTGGCTGTACCAAGTGGCATGGACTCCGCTGGATACGCTGCCGCCTGCACCGGAACCGTCGGCGAAACTGTGGCTGATTCTGGGTGATCGTCACGATCACCAACCGATTGAGGCCCAGTTCAAAAACGCCCAACGTGTGTACCTGGGCCAAAGCAACCACTTTCCGACGAACGCCCCGTGGGAGGTGAGCGCGGACGCACTGGATAACTTGTTTACCCATGTGGGTAGCCAAAACCTGGCAGGCATTCTGTATCTGTGCCCGCCTGGTGAAGATCCGGAGGATCTGGATGAGATTCAGAAACAAACTTCCGGCTTTGCGTTGCAACTGATTCAGACCCTGTATCAGCAGAAAATCGCAGTGCCGTGTTGGTTTGTTACCCATCAAAGCCAGCGTGTGCTGGAAACGGACGCGGTGACGGGTTTTGCCCAAGGTGGTCTGTGGGGTTTGGCGCAAGCGATTGCACTGGAACATCCGGAACTGTGGGGTGGTATCATTGACGTGGATGATAGCCTGCCGAACTTCGCGCAGATTTGTCAGCAACGTCAGGTTCAGCAACTGGCTGTCCGTCACCAGAAACTGTATGGTGCGCAACTGAAGAAGCAGCCGAGCCTGCCGCAGAAGAATCTGCAGATCCAACCTCAACAGACCTACCTGGTCACGGGCGGTTTGGGTGCAATCGGTCGTAAGATTGCGCAGTGGCTGGCGGCTGCGGGTGCTGAGAAAGTTATCCTGGTTAGCCGTCGTGCACCGGCAGCGGATCAACAAACCTTGCCGACCAACGCCGTGGTGTACCCGTGCGATCTGGCGGATGCGGCGCAGGTTGCGAAACTGTTCCAAACCTATCCGCACATTAAGGGTATCTTTCATGCAGCCGGTACGCTGGCTGACGGTTTGCTGCAACAGCAAACCTGGCAGAAATTCCAGACTGTCGCTGCGGCGAAGATGAAGGGCACCTGGCACCTGCATCGCCACTCTCAGAAGTTGGACTTGGATTTCTTTGTTTTGTTTTCGTCTGTTGCGGGTGTGCTGGGTAGCCCTGGTCAAGGCAATTACGCGGCAGCCAACCGTGGCATGGCCGCCATCGCTCAGTACCGCCAGGCTCAAGGTCTGCCGGCACTGGCGATTCACTGGGGCCCTTGGGCGGAAGGTGGTATGGCAAACAGCTTGAGCAACCAAAATCTGGCATGGTTGCCTCCGCCGCAGGGCTTGACCATTCTGGAAAAAGTTTTGGGTGCCCAAGGCGAAATGGGCGTGTTCAAACCGGACTGGCAGAACTTGGCCAAACAATTCCCGGAGTTCGCGAAAACCCATTACTTTGCGGCGGTCATTCCGAGCGCTGAAGCGGTTCCACCGACCGCATCTATCTTCGACAAGCTGATCAATCTGGAAGCGAGCCAGCGCGCAGATTACCTGCTGGACTATCTGCGTAGATCTGTGGCACAAATTCTGAAACTGGAAATTGAGCAGATTCAGAGCCACGACTCCCTGCTGGATCTGGGTATGGATAGCCTGATGATCATGGAGGCGATTGCGTCCCTGAAACAAGACCTGCAACTGATGCTGTATCCGCGTGAGATTTACGAGCGTCCGCGTCTGGATGTTCTGACTGCTTACTTGGCCGCTGAGTTTACCAAAGCGCATGATTCTGAAGCAGCTACCGCCGCAGCTGCGATCCCTAGCCAGAGCCTGAGCGTCAAAACCAAAAAGCAATGGCAGAAACCGGATCATAAGAACCCGAATCCGATTGCGTTCATCCTGAGCAGCCCGCGTAGCGGTAGCACCCTGCTGCGCGTGATGCTGGCCGGTCACCCGGGTCTGTATTCCCCACCGGAACTGCACCTGCTGCCGTTTGAAACGATGGGTGACCGCCACCAGGAACTGGGTCTGTCTCATCTGGGCGAGGGTCTGCAACGTGCCCTGATGGACTTGGAAAATCTGACGCCGGAAGCATCCCAGGCAAAGGTGAACCAATGGGTGAAGGCGAATACGCCGATTGCAGACATCTACGCATACCTGCAACGTCAAGCCGAGCAACGTCTGCTGATTGACAAAAGCCCGAGCTATGGCAGCGACCGCCACATTCTGGATCACAGCGAGATCCTGTTCGATCAGGCGAAATACATCCACCTGGTTCGCCATCCTTATGCGGTCATTGAGAGCTTTACCCGCCTGCGTATGGACAAGCTGCTGGGTGCAGAGCAACAGAATCCGTATGCGCTGGCGGAAAGCATTTGGCGTACCTCGAATCGCAACATTCTGGACTTGGGTCGTACCGTCGGCGCTGACCGCTACCTGCAAGTCATCTACGAGGATCTGGTGCGTGACCCGCGTAAAGTTCTGACCAACATTTGTGATTTTCTGGGTGTCGATTTCGACGAGGCACTGCTGAATCCGTACTCCGGCGACCGCCTGACCGACGGCCTGCACCAGCAAAGCATGGGTGTGGGTGACCCGAACTTCTTGCAGCACAAGACCATTGATCCGGCGCTAGCGGACAAATGGCGTAGCATTACCCTGCCGGCTGCTCTGCAACTGGATACGATTCAACTGGCCGAAACCTTCGCATACGACCTGCCGCAGGAGCCGCAGTTGACGCCGCAGACCCAATCTTTGCCATCGATGGTCGAACGTTTCGTCACGGTTCGCGGCCTGGAAACCTGTCTGTGCGAGTGGGGTGATCGCCATCAACCTCTGGTCTTGCTGTTGCACGGTATCCTGGAGCAAGGCGCGTCTTGGCAGTTGATCGCGCCTCAACTGGCAGCGCAGGGCTATTGGGTCGTCGCTCCGGATCTGCGCGGTCACGGTAAATCTGCGCACGCGCAGTCTTATAGCATGCTGGATTTTCTGGCCGATGTGGACGCGCTGGCCAAACAGTTGGGCGACCGTCCGTTCACCTTGGTTGGTCACAGCATGGGTTCCATCATTGGCGCAATGTATGCTGGCATTCGTCAAACCCAGGTTGAAAAACTGATTCTGGTCGAAACCATCGTCCCGAATGATATTGATGATGCCGAAACCGGCAATCACCTGACCACCCATCTGGATTACCTGGCAGCCCCTCCGCAGCACCCGATCTTTCCGAGCCTGGAAGTTGCGGCTCGTCGTCTGCGCCAAGCCACCCCGCAGTTGCCGAAAGACCTGTCTGCATTTCTGACGCAACGTTCCACGAAGAGCGTCGAGAAGGGTGTGCAGTGGCGCTGGGATGCCTTCTTGCGCACCCGTGCAGGTATCGAGTTTAACGGTATCAGCCGTCGCCGTTATCTGGCGCTGCTGAAAGATATCCAGGCCCCAATTACTTTGATTTACGGTGATCAGTCTGAGTTCAATCGCCCAGCAGACCTGCAAGCGATCCAGGCGGCACTGCCGCAAGCGCAACGCCTGACGGTTGCTGGCGGTCACAACTTGCACTTTGAGAATCCGCAGGCCATCGCCCAGATTGTCTATCAGCAGTTGCAGACACCGGTTCCGAAAACCCAAGGTTTGCACCATCACCACCATCATAGCGCCTGGAGCCACCCGCAGTTTGAAAAGTAA SEQ ID NO: 28 nonA_mycANucleotide SequenceThe Interior Acyl Binding Pocket-encoding sequence is underlined.ATGGCAAGCTGGTCCCACCCGCAATTCGAGAAAGAAGTACATCACCATCACCATCATGGCGCAGTGGGCCAGTTTGCGAACTTTGTAGACCTGTTGCAATACCGTGCCAAGCTGCAAGCACGTAAGACCGTCTTTAGCTTCCTGGCGGACGGCGAAGCGGAGAGCGCCGCTCTGACCTATGGTGAGCTGGATCAAAAGGCGCAGGCAATCGCGGCGTTCCTGCAAGCAAATCAGGCACAAGGCCAACGTGCATTGCTGCTGTATCCGCCAGGTCTGGAGTTCATCGGTGCCTTCCTGGGTTGTCTGTATGCGGGTGTCGTCGCGGTTCCGGCATATCCTCCGCGTCCGAACAAGTCCTTCGACCGTTTGCACTCCATCATTCAGGACGCCCAAGCGAAGTTTGCACTGACGACGACCGAGTTGAAGGATAAGATTGCAGACCGTCTGGAAGCGCTGGAGGGTACGGACTTCCATTGCCTGGCGACCGACCAAGTCGAGCTGATCAGCGGCAAAAACTGGCAAAAGCCGAATATCTCCGGTACGGATCTGGCGTTTCTGCAATACACCAGCGGCAGCACGGGTGATCCAAAAGGCGTGATGGTCAGCCACCATAACCTGATTCACAATAGCGGTCTGATTCGCAACGCGCTGGCGATTGATCTGAAAGATACCCTGCTGTCTTGGATGCCGTTGACTCACGATATGGGTTTGATTGCGTGCCATCTGGTTCCGGCGCTGGCGGGCATTAACCAGAATTTGATGCCGACTGAACTGTTCATTCGTCGCCCGATTCTGTGGATGAAGAAAGCTCACGAACATAAAGCGTCTATTCTGTCTAGCCCGAATTTCGGTTACAACTACTTTCTGAAATTCCTGAAAGACAACAAAAGCTACGATTGGGATCTGTCCCATATTCGCGTTATCGCGAACGGTGCAGAGCCGATTCGCGCTGTCACGCTGGAAAACTTTGCGAAAACGTTCGCAACCGCGGGTTTCCAGAAATCGGCCTTCTACCCTTGTTACGGTATGGCGGAAACCACCCTGATCGTGAGCGGTGGCAATGGCCGTGCCCAACTGCCACAGGAGATCATCGTTAGCAAGCAGGGCATTGAGGCGAACCAAGTGCGTCCGGCTCAAGGCACGGAAACGACCGTGACCCTGGTGGGTAGCGGTGAGGTCATTGGTGACCAGATCGTTAAGATCGTTGACCCTCAAGCGCTGACCGAGTGCACCGTCGGTGAAATTGGCGAGGTGTGGGTTAAAGGTGAAAGCGTTGCTCAGGGCTACTGGCAGAAGCCGGACTTGACGCAGCAGCAGTTCCAGGGTAACGTGGGTGCCGAAACGGGTTTCCTGCGCACCGGCGATCTGGGTTTCCTGCAAGGCGGCGAGCTGTATATCACCGGCCGTCTGAAGGATCTGCTGATCATTCGTGGCCGTAATCACTATCCTCAGGACATTGAGCTGACCGTGGAAGTTGCTCACCCAGCCCTGCGTCAGGGCGCAGGTGCCGCGGTGAGCGTGGACGTTAATGGTGAAGAACAACTGGTGATCGTTCAAGAGGTTGAGCGTAAGTACGCACGCAAGCTGAATGTGGCAGCAGTCGCTCAGGCCATCCGTGGTGCGATTGCGGCAGAGCACCAGTTGCAGCCGCAGGCGATCTGCTTTATCAAACCGGGCAGCATCCCGAAAACTAGCAGCGGCAAAATCCGTCGTCACGCATGTAAGGCCGGTTTTCTGGACGGAAGCTTGGCGGTTGTTGGTGAGTGGCAACCGAGCCATCAGAAAGAGGGCAAAGGTATTGGTACCCAGGCAGTGACCCCGAGCACCACGACGTCCACCAACTTTCCGCTGCCGGATCAACACCAGCAACAGATCGAGGCGTGGCTGAAGGACAACATCGCGCACCGCCTGGGTATTACGCCGCAGCAGTTGGATGAAACGGAACCGTTCGCTTCTTACGGTCTGGACAGCGTTCAAGCAGTCCAGGTCACCGCAGACCTGGAGGACTGGCTGGGCCGCAAGCTGGACCCGACTCTGGCCTATGATTACCCGACCATTCGCACGCTGGCGCAATTCCTGGTTCAGGGCAACCAGGCCTTGGAGAAAATCCCGCAAGTTCCAAAGATTCAGGGTAAAGAGATTGCGGTGGTGGGCCTGAGCTGCCGCTTTCCGCAGGCGGACAATCCGGAGGCGTTCTGGGAACTGTTGCGCAATGGCAAGGATGGCGTGCGTCCGCTGAAAACCCGTTGGGCCACTGGTGAGTGGGGTGGTTTCCTGGAGGATATCGACCAGTTTGAGCCGCAGTTCTTTGGTATTAGCCCGCGTGAGGCGGAGCAAATGGACCCGCAACAGCGTCTGCTGCTGGAGGTCACCTGGGAGGCACTGGAGCGTGCGAATATCCCTGCCGAATCCCTGCGTCACAGCCAGACCGGCGTCTTTGTGGGCATTAGCAACAGCGATTACGCACAACTGCAAGTGCGTGAGAACAACCCGATCAATCCGTACATGGGTACTGGTAACGCACATAGCATCGCGGCGAATCGTCTGAGCTACTTTCTGGATCTGCGCGGTGTCTCCCTGAGCATTGATACCGCGTGTTCTAGCAGCCTGGTCGCAGTTCATCTGGCGTGCCAAAGCCTGATTAACGGCGAGAGCGAGCTGGCGATTGCTGCGGGTGTTAATCTGATTCTGACCCCGGATGTCACGCAAACCTTTACCCAAGCGGGTATGATGAGCAAGACGGGCCGTTGCCAGACGTTTGATGCGGAGGCGGACGGCTACGTGCGCGGTGAAGGCTGCGGCGTTGTTCTGCTGAAACCGCTGGCTCAGGCGGAGCGTGATGGCGACAATATCCTGGCGGTCATCCACGGTAGCGCGGTTAACCAGGACGGTCGCAGCAATGGTCTGACTGCGCCGAACGGCCGCTCTCAGCAAGCGGTTATCCGTCAGGCCCTGGCGCAGGCGGGCATCACCGCGGCAGACCTGGCGTATTTGGAAGCGCATGGTACGGGCACCCCGCTGGGCGACCCGATTGAAATCAACAGCTTGAAAGCAGTGCTGCAAACCGCCCAGCGCGAGCAACCGTGCGTTGTGGGCAGCGTCAAGACGAACATTGGCCACCTGGAGGCAGCAGCGGGTATTGCAGGTCTGATCAAGGTGATTCTGTCCCTGGAGCACGGCATGATTCCGCAACACCTGCACTTTAAGCAACTGAATCCGCGCATCGACCTGGACGGCCTGGTTACCATCGCGAGCAAAGACCAGCCGTGGTCGGGTGGTAGCCAGAAGCGTTTCGCCGGTGTCAGCAGCTTTGGTTTTGGCGGTACGAATGCTCACGTGATTGTTGGTGATTATGCCCAGCAAAAGTCCCCGCTGGCTCCGCCTGCGACCCAAGACCGTCCTTGGCATCTGCTGACTCTGAGCGCGAAGAACGCACAAGCGTTGAACGCGTTGCAAAAGAGCTATGGTGACTACCTGGCGCAACATCCGAGCGTTGACCCTCGCGATCTGTGCCTGAGCGCTAACACTGGTCGCTCTCCGCTGAAAGAACGCCGCTTCTTCGTGTTCAAGCAGGTTGCCGACTTGCAACAAACCCTGAATCAGGACTTTCTGGCGCAGCCGAGGCTGAGCAGCCCAGCCAAGATTGCGTTCCTGTTCACGGGTCAGGGCAGCCAGTACTACGGTATGGGCCAGCAACTGTATCAGACGTCCCCGGTTTTCCGTCAAGTCCTGGATGAATGCGACCGTCTGTGGCAGACGTACAGCCCGGAGGCACCGGCGCTGACCGATCTGCTGTACGGCAATCATAATCCTGACCTGGTTCATGAAACGGTTTACACGCAACCGCTGCTGTTCGCGGTGGAGTATGCTATCGCGCAGTTGTGGTTGAGCTGGGGCGTTACTCCGGATTTCTGCATGGGTCATAGCGTCGGTGAGTATGTGGCGGCCTGCCTGGCGGGTGTGTTTAGCCTGGCGGATGGCATGAAACTGATTACCGCGCGTGGTAAACTGATGCATGCACTGCCGAGCAATGGCAGCATGGCGGCTGTGTTTGCGGACAAAACCGTTATCAAGCCGTATCTGAGCGAACACCTGACCGTCGGCGCAGAAAATGGCAGCCACCTGGTTCTGAGCGGTAAGACCCCTTGTCTGGAAGCATCCATCCACAAACTGCAAAGCCAGGGCATCAAAACCAAGCCTCTGAAAGTCTCCCATGCGTTCCACTCGCCGCTGATGGCGCCGATGCTGGCGGAATTTCGTGAGATCGCCGAACAGATTACGTTCCATCCGCCACGTATCCCGCTGATTAGCAACGTGACGGGTGGTCAAATCGAGGCCGAGATCGCGCAAGCAGACTATTGGGTTAAACATGTTAGCCAGCCGGTGAAGTTCGTTCAGAGCATTCAGACCCTGGCCCAAGCGGGTGTGAATGTGTACCTGGAAATCGGTGTTAAACCAGTCCTGCTGTCTATGGGTCGCCACTGTCTGGCAGAGCAGGAAGCGGTTTGGCTGCCGAGCCTGCGTCCACATAGCGAGCCTTGGCCGGAAATCTTGACTAGTCTGGGCAAACTGTACGAGCAAGGTCTGAATATCGACTGGCAAACGGTTGAAGCCGGTGATCGCCGTCGTAAGCTGATTTTGCCGACCTACCCGTTCCAGCGTCAGCGTTATTGGTTCAACCAAGGTAGCTGGCAAACCGTCGAAACTGAGAGCGTGAATCCAGGCCCGGACGACCTGAATGACTGGCTGTACCAAGTGGCATGGACTCCGCTGGATACGCTGCCGCCTGCACCGGAACCGTCGGCGAAACTGTGGCTGATTCTGGGTGATCGTCACGATCACCAACCGATTGAGGCCCAGTTCAAAAACGCCCAACGTGTGTACCTGGGCCAAAGCAACCACTTTCCGACGAACGCCCCGTGGGAGGTGAGCGCGGACGCACTGGATAACTTGTTTACCCATGTGGGTAGCCAAAACCTGGCAGGCATTCTGTATCTGTGCCCGCCTGGTGAAGATCCGGAGGATCTGGATGAGATTCAGAAACAAACTTCCGGCTTTGCGTTGCAACTGATTCAGACCCTGTATCAGCAGAAAATCGCAGTGCCGTGTTGGTTTGTTACCCATCAAAGCCAGCGTGTGCTGGAAACGGACGCGGTGACGGGTTTTGCCCAAGGTGGTCTGTGGGGTTTGGCGCAAGCGATTGCACTGGAACATCCGGAACTGTGGGGTGGTATCATTGACGTGGATGATAGCCTGCCGAACTTCGCGCAGATTTGTCAGCAACGTCAGGTTCAGCAACTGGCTGTCCGTCACCAGAAACTGTATGGTGCGCAACTGAAGAAGCAGCCGAGCCTGCCGCAGAAGAATCTGCAGATCCAACCTCAACAGACCTACCTGGTCACGGGCGGTTTGGGTGCAATCGGTCGTAAGATTGCGCAGTGGCTGGCGGCTGCGGGTGCTGAGAAAGTTATCCTGGTTAGCCGTCGTGCACCGGCAGCGGATCAACAAACCTTGCCGACCAACGCCGTGGTGTACCCGTGCGATCTGGCGGATGCGGCGCAGGTTGCGAAACTGTTCCAAACCTATCCGCACATTAAGGGTATCTTTCATGCAGCCGGTACGCTGGCTGACGGTTTGCTGCAACAGCAAACCTGGCAGAAATTCCAGACTGTCGCTGCGGCGAAGATGAAGGGCACCTGGCACCTGCATCGCCACTCTCAGAAGTTGGACTTGGATTTCTTTGTTTTGTTTTCGTCTGTTGCGGGTGTGCTGGGTAGCCCTGGTCAAGGCAATTACGCGGCAGCCAACCGTGGCATGGCCGCCATCGCTCAGTACCGCCAGGCTCAAGGTCTGCCGGCACTGGCGATTCACTGGGGCCCTTGGGCGGAAGGTGGTATGGCAAACAGCTTGAGCAACCAAAATCTGGCATGGTTGCCTCCGCCGCAGGGCTTGACCATTCTGGAAAAAGTTTTGGGTGCCCAAGGCGAAATGGGCGTGTTCAAACCGGACTGGCAGAACTTGGCCAAACAATTCCCGGAGTTCGCGAAAACCCATTACTTTGCGGCGGTCATTCCGAGCGCTGAAGCGGTTCCACCGACCGCATCTATCTTCGACAAGCTGATCAATCTGGAAGCGAGCCAGCGCGCAGATTACCTGCTGGACTATCTGCGTAGATCTGTGGCACAAATTCTGAAACTGGAAATTGAGCAGATTCAGAGCCACGACTCCCTGCTGGATCTGGGTATGGATAGCCTGATGATCATGGAGGCGATTGCGTCCCTGAAACAAGACCTGCAACTGATGCTGTATCCGCGTGAGATTTACGAGCGTCCGCGTCTGGATGTTCTGACTGCTTACTTGGCCGCTGAGTTTACCAAAGCGCATGATTCTGAAGCAGCTACCGCCGCAGCTGCGATCCCTAGCCAGAGCCTGAGCGTCAAAACCAAAAAGCAATGGCAGAAACCGGATCATAAGAACCCGAATCCGATTGCGTTCATCCTGAGCAGCCCGCGTAGCGGTAGCACCCTGCTGCGCGTGATGCTGGCCGGTCACCCGGGTCTGTATTCCCCACCGGAACTGCACCTGCTGCCGTTTGAAACGATGGGTGACCGCCACCAGGAACTGGGTCTGTCTCATCTGGGCGAGGGTCTGCAACGTGCCCTGATGGACTTGGAAAATCTGACGCCGGAAGCATCCCAGGCAAAGGTGAACCAATGGGTGAAGGCGAATACGCCGATTGCAGACATCTACGCATACCTGCAACGTCAAGCCGAGCAACGTCTGCTGATTGACAAAAGCCCGAGCTATGGCAGCGACCGCCACATTCTGGATCACAGCGAGATCCTGTTCGATCAGGCGAAATACATCCACCTGGTTCGCCATCCTTATGCGGTCATTGAGAGCTTTACCCGCCTGCGTATGGACAAGCTGCTGGGTGCAGAGCAACAGAATCCGTATGCGCTGGCGGAAAGCATTTGGCGTACCTCGAATCGCAACATTCTGGACTTGGGTCGTACCGTCGGCGCTGACCGCTACCTGCAAGTCATCTACGAGGATCTGGTGCGTGACCCGCGTAAAGTTCTGACCAACATTTGTGATTTTCTGGGTGTCGATTTCGACGAGGCACTGCTGAATCCGTACTCCGGCGACCGCCTGACCGACGGCCTGCACCAGCAAAGCATGGGTGTGGGTGACCCGAACTTCTTGCAGCACAAGACCATTGATCCGGCGCTAGCGGACAAATGGCGTAGCATTACCCTGCCGGCTGCTCTGCAACTGGATACGATTCAACTGGCCGAAACCTTCGCATACGACCTGCCGCAGGAGCCGCAGTTGACGCCGCAGACCCAATCTTTGCCATCGATGGTCGAACGTTTCGTCACGGTTCGCGGCCTGGAAACCTGTCTGTGCGAGTGGGGTGATCGCCATCAACCTCTGGTCTTGCTGTTGCACGGTATCCTGGAGCAAGGCGCGTCTTGGCAGTTGATCGCGCCTCAACTGGCAGCGCAGGGCTATTGGGTCGTCGCTCCGGATCTGCGCGGTCACGGTAAATCTGCGCACGCGCAGTCTTATAGCATGCTGGATTTTCTGGCCGATGTGGACGCGCTGGCCAAACAGTTGGGCGACCGTCCGTTCACCTTGGTTGGTCACAGCATGGGTTCCATCATTGGCGCAATGTATGCTGGCATTCGTCAAACCCAGGTTGAAAAACTGATTCTGGTCGAAACCATCGTCCCGAATGATATTGATGATGCCGAAACCGGCAATCACCTGACCACCCATCTGGATTACCTGGCAGCCCCTCCGCAGCACCCGATCTTTCCGAGCCTGGAAGTTGCGGCTCGTCGTCTGCGCCAAGCCACCCCGCAGTTGCCGAAAGACCTGTCTGCATTTCTGACGCAACGTTCCACGAAGAGCGTCGAGAAGGGTGTGCAGTGGCGCTGGGATGCCTTCTTGCGCACCCGTGCAGGTATCGAGTTTAACGGTATCAGCCGTCGCCGTTATCTGGCGCTGCTGAAAGATATCCAGGCCCCAATTACTTTGATTTACGGTGATCAGTCTGAGTTCAATCGCCCAGCAGACCTGCAAGCGATCCAGGCGGCACTGCCGCAAGCGCAACGCCTGACGGTTGCTGGCGGTCACAACTTGCACTTTGAGAATCCGCAGGCCATCGCCCAGATTGTCTATCAGCAGTTGCAGACACCGGTTCCGAAAACCCAAGGTTTGCACCATCACCACCATCATAGCGCCTGGAGCCACCCGCAGTTTGAAAAGTAA SEQ ID NO: 29 NonA_dptEAmino Acid SequenceThe Interior Acyl Binding Pocket sequence is underlined.MASWSHPQFEKEVHHHHHHGAVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLLAEACELTAATPMGGWLPMYHDMGLLGTLTPALYLGTTCVLMSSTAFIKRPHLWLRTIDRFGLVWSSAPDFAYDMCLKRVTDEQIAGLDLSRWRWAGNGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQGLHHHHHHSAWSHPQFEKSEQ ID NO: 30 NonA_safB Amino Acid SequenceThe Interior Acyl Binding Pocket sequence is underlined.MASWSHPQFEKEVHHHHHHGAVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLIFTSFHMNDETIIFSWLPPHHDMGLIGCILTPIYGGIQAIMMSPFSFLQNPLSWLKHITKYKATISGSPNFAYDYCVKRIREEKKEGLDLSSWVTAFNGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQGLHHHHHHSAWSHPQFEKSEQ ID NO: 31 NonA_mycA Amino Acid SequenceThe Interior Acyl Binding Pocket sequence is underlined.MASWSHPQFEKEVHHHHHHGAVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAIAAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTELKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNSGLIRNALAIDLKDTLLSWMPLTHDMGLIACHLVPALAGINQNLMPTELFIRRPILWMKKAHEHKASILSSPNFGYNYFLKFLKDNKSYDWDLSHIRVIANGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIVSGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWVKGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTVEVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGSIPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLKDNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQALEKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFFGISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGNAHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGMMSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAVIRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGLIKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQQKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFKQVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPALTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKLITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKPLKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQTLAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGDRRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGDRHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQKQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDDSLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGAEKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQTVAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGPWAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAVPPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQLMLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIAFILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPYSGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQSLPSMVERFVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIVYQQLQTPVPKTQGLHHHHHHSAWSHPQFEKSEQ ID NO: 32 PCR primer A2265 Forward Primer SacI Nucleotide SequenceggGAGCTCaaggaattatagttatgcgcaaaccctggttaga SEQ ID NO: 33 PCR primerA2265 Reverse Primer SacI Nucleotide SequenceggCCTGCAGGttatagggactggatcgccagttttttctgct SEQ ID NO: 34 NucleotideInterior Acyl Binding Pocket of dptECTCGCCGAGGCCTGCGAGCTGACCGCCGCCACTCCCATGGGCGGCTGGCTGCCCATGTACCACGACATGGGGCTCCTGGGCACGCTGACACCGGCCCTGTACCTCGGCACCACGTGCGTGCTGATGAGCTCCACGGCATTCATCAAACGGCCGCACCTGTGGCTACGGACCATCGACCGGTTCGGCCTGGTCTGGTCGTCGGCTCCCGACTTCGCGTACGACATGTGTCTGAAGCGCGTCACCGACGAGCAGATCGCCGGGCTGGACCTGTCCCGCTGGCGGTGGGCCGGCAACSEQ ID NO: 35 Nucleotide Interior Acyl Binding Pocket of safBATTTTTACCTCTTTTCATATGAATGATGAAACCATTATTTTCAGCTGGCTGCCCCCACATCATGATATGGGTTTGATTGGCTGCATTCTGACCCCCATCTATGGTGGAATTCAGGCAATCATGATGTCCCCTTTCTCATTTTTACAAAACCCGCTTTCCTGGTTAAAACATATTACCAAATACAAAGCAACTATCAGTGGAAGCCCTAACTTCGCTTACGATTATTGTGTCAAACGAATCAGGGAAGAAAAAAAAGAAGGGCTGGATTTAAGTTCATGGGTGACTGCTTTCAACSEQ ID NO: 36 Nucleotide Interior Acyl Binding Pocket of mycAATCCGGAATGCGCTGGCTATCGACTTAAAAGATACTCTTTTATCTTGGATGCCCTTAACCCATGACATGGGGCTCATAGCTTGCCACCTTGTTCCTGCCTTAGCCGGAATCAATCAAAATTTAATGCCGACAGAATTATTTATTCGAAGACCTATTCTCTGGATGAAAAAAGCTCATGAACATAAAGCCAGCATTCTATCCTCACCTAATTTTGGATACAATTACTTTCTTAAATTTCTGAAAGACAATAAAAGTTACGACTGGGATTTATCCCATATCAGGGTCATTGCAAACSEQ ID NO: 37 Nucleotide A2265 Synechococcus sp. PCC 7002locus: SYNPCC7002_A2265 Accession No: NC_010475.1: 2037569 . . . 20385521 gtgcgcaaac cctggttaga acttcccttg gcgatttttt cctttggctt ttataaagtc 61aacaaatttc tgattgggaa tctctacact ttgtatttag cgctgaataa aaaaaatgct 121aaggaatggc gcattattgg agaaaaatcc ctccagaaat tcctgagttt acccgtttta 181atgaccaaag cgccccggtg gaatacccac gccattatcg gcaccctggg accactctct 241gtagaaaaag aactcaccat taacctcgaa acgattcgtc aatccacgga agcttgggtc 301ggttgcatct atgactttcc gggctatcgc acggtgttaa atttcacgca actcaccgat 361gaccccaacc aaacagaact caaaattttc ttacctaaag ggaaatatac cgtcgggtta 421cgttactacc atcccaaggt aaatcctcgc tttccggtcg ttaaaacaga tctaaatcta 481accgtgccga ctttggttgt ttcgccccaa aacaacgact tttatcaagc cctggcccag 541aaaacaaacc tttattttcg tctgcttcac tactacattt ttacgctatt taaatttcgc 601gatgtcttac ccgctgcttt tgtgaaagga gaattcctcc ctgtcggcgc caccgatact 661caattttttt acggcgcttt agaagcagca gaaaacttag agattaccat cccagccccc 721tggcttcaga cctttgattt ttatctcacc ttctataacc gcgccagttt tcccctacgt 781tggcaaaaaa tcaccgaagc gatgatctgt gatcccctgg gagaaaaagg ctattaccta 841attcggatgc ggccccgtac tcaggacgcc gaggcacaat taccaacggt tagaggagaa 901gaaacccagg tcacgcccca gcagaaaaaa ctggcgatcc agtccctata a SEQ ID NO: 38Amino Acid Synechococcus sp. PCC 7002 aoa locus: SYNPCC7002_A2265Accession No: YP_001735499.1 1mrkpwlelpl aifsfgfykv nkflignlyt lylalnkkna kewriigeks lqkflslpvl 61mtkaprwnth aiigtlgpls vekeltinle tirqsteawv gciydfpgyr tvinftqltd 121dpnqtelkif lpkgkytvgl ryyhpkvnpr fpvvktdlnl tvptlvvspq nndfyqalaq 181ktnlyfrllh yyiftlfkfr dvlpaafvkg eflpvgatdt qffygaleaa enleitipap 241wlqtfdfylt fynrasfplr wqkiteamic dplgekgyyl irmrprtqda eaqlptvrge 301etqvtpqqkk laiqsl

What is claimed is:
 1. An isolated or recombinant chimeric NonA alkenesynthase comprising a heterologous acyl binding pocket.
 2. The alkenesynthase of claim 1, wherein said heterologous acyl binding pocketcomprises a polypeptide sequence selected from the group consisting of:a. SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16; and b. a polypeptidesequence at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, atleast 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQID NO: 8, SEQ ID NO: 12, or SEQ ID NO:
 16. 3. An isolated or recombinantpolynucleotide encoding the heterologous acyl binding pocket of claim 1,comprising or consisting of: a. SEQ ID NO: 35, SEQ ID NO: 36, or SEQ IDNO: 34; b. a nucleotide sequence that is a degenerate variant of SEQ IDNO: 35, SEQ ID NO: 36, or SEQ ID NO: 34; c. a nucleic acid sequence atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99% or at least 99.9%identical to SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34; d. anucleic acid sequence that encodes a polypeptide at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, atleast 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least99.8% or at least 99.9% identical to SEQ ID NO: 8, SEQ ID NO: 12, or SEQID NO: 16; or e. a nucleic acid sequence that hybridizes under stringentconditions to SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO:
 34. 4. Anisolated or recombinant polypeptide comprising or consisting of: a. SEQID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29; or b. a polypeptide sequenceat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%,at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 30, SEQID NO: 31, or SEQ ID NO:
 29. 5. An isolated or recombinantpolynucleotide comprising or consisting of: a. SEQ ID NO: 27, SEQ ID NO:28, or SEQ ID NO: 26; b. a nucleotide sequence that is a degeneratevariant of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26; c. annucleotide sequence at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 90%, at least 95%, at least 98%, at least99% or at least 99.9% identical to SEQ ID NO: 27, SEQ ID NO: 28, or SEQID NO: 26; d. a nucleic acid sequence that encodes a polypeptide havingthe amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO:29; e. a nucleic acid sequence that encodes a polypeptide at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%%, at least 99.1%, at least 99.2%, at least99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%,at least 99.8% or at least 99.9% identical to SEQ ID NO: 30, SEQ ID NO:31, or SEQ ID NO: 29; or f. a nucleic acid sequence that hybridizesunder stringent conditions to a nucleic acid sequence that encodes apolypeptide having the amino acid sequence of SEQ ID NO: 27, SEQ ID NO:28, or SEQ ID NO:
 26. 6. The isolated or recombinant polynucleotide ofclaim 3 or claim 5, wherein the nucleic acid sequence is operably linkedto one or more expression control sequences.
 7. A vector comprising theisolated or recombinant polynucleotide of claim 3 or claim
 5. 8. Afusion protein comprising the polypeptide of claim 2 or claim 4 fused toa heterologous amino acid sequence.
 9. A host cell comprising theisolated or recombinant polynucleotide of claim 3 or claim
 5. 10. Thehost cell of claim 9, wherein the host cell is selected from the groupconsisting of prokaryotes, eukaryotes, yeasts, filamentous fungi,protozoa, algae and synthetic cells.
 11. The host cell of claim 9,wherein the host cell is a photoautotroph.
 12. The host cell of claim 9,wherein the host cell is E. coli.
 13. The host cell of claims 9 whereinthe host cell produces carbon-based products of interest.
 14. Anisolated antibody or antigen-binding fragment or derivative thereofwhich binds selectively to the polypeptide of claim 3 or claim
 5. 15. Amethod for producing a carbon-based product of interest comprising: a.culturing a host cell to produce the carbon-based product of interest,wherein said host cell comprises an engineered chimeric alkene synthasecomprising a heterologous acyl binding pocket; and b. isolating thecarbon-based product of interest.
 16. The method of claim 15, whereinthe chimeric alkene synthase is derived from NonA.
 17. The method ofclaim 16, wherein said NonA comprises SEQ ID NO:
 2. 18. The method ofclaim 16, wherein said NonA comprises SEQ ID NO:
 24. 19. The method ofclaim 15, wherein the heterologous acyl binding pocket comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 8,SEQ ID NO: 12, and SEQ ID NO:
 16. 20. The method of claim 15, whereinsaid chimeric alkene synthase selectively synthesizes one or morealkenes with specific chain lengths.